Small molecule agonists and antagonists of nr2f6 activity in animals

ABSTRACT

The present technology is directed to modulators of nuclear receptor activity, specifically to the modulation of NR2F6 activity and NR2F6 utilizing compounds, and the immune modulation and modulation of cancer stem cell activity through administration of compounds described herein to animals.

BACKGROUND

The present technology relates to agonists and antagonists of nuclearreceptor activity, specifically to the modulation of NR2F6 activity andNR2F6 utilizing compounds, and the immune modulation and modulation ofcancer stem cell activity through administration of compounds describedherein.

Many drugs administered to treat diseases or conditions are targetedagainst differences between a diseased cell and a normal cell. T cellsof the immune system are known to recognize and interact with specificmolecules through receptors (e.g., a T cell receptor in complex with aCD3 dimer) which, upon recognition or interaction with these molecules,result in the activation of the T cell to perform various immuneactivities. Innate immune cells are cells of the immune system that areknown to be activated by one or more agents (e.g., allergens, chemicalsproduced upon injury (e.g., opioids and alcohols), polymvxins,crosslinked IgE, crosslinked complement proteins, cytokines produced byT cells or other immune cells (e.g., interferon-γ), DAMPs, or PAMPs)that activate downstream signaling pathway(s) in the innate immune celland result in the activation of one or more immune activities of theinnate immune cell.

Both T cells and innate immune cells play a role in a mammal's immunedefense. For example, the immune activities of an innate immune cell canprotect a mammal against infectious diseases. The immune activities of aT cell can protect a mammal against, for example, infectious diseasesand cancer.

Adoptive cell therapy is a method of treatment that includes harvestingone or more different types of immune cells from a mammal, culturingand/or manipulating the harvested immune cells ex vivo, andadministering the cultured and/or manipulated immune cells back to themammal. The manipulating of a harvested immune cell ex vivo can includeintroducing a recombinant nucleic acid into the immune cell.

Molecularly targeted therapeutics represent a new approach todiscovering anti-cancer drugs. Using this approach, small molecules aredesigned to inhibit directly the very oncogenetic proteins that aremutated or overexpressed in specific tumor cell types. By targetingspecific molecular defects or conditions found within tumor cells, thisapproach can yield therapies tailored to each tumor's genetic makeup. Acomplementary strategy involves searching for genotype-selectiveanti-tumor agents that become lethal to tumor cells only in the presenceof specific oncoproteins or only in the absence of specific tumoroppressors. Such genotype-selective compounds might target oncoproteinsdirectly, or target other critical proteins involved inoncoprotein-linked signaling networks.

The immune system is comprised of activatory and inhibitory mechanismsthat can allow for control of immune responses and subsequent inhibitionof responses after clearance of the immune target. The central eventstimulating immune responses is the antigen-specific activation of naiveCD4⁺ T cells subsequent to binding antigen presenting cell MHCcontaining antigenic peptide. The CD4⁺ T cell, also known as the “helperT cell,” helps to coordinate the activation of the adaptive immuneresponse, playing a role in the stimulation of cytotoxic CD8+ T cells,whose role includes destroying host cells affected by cancer, viruses,and intracellular bacteria, as well as stimulating B cell maturation toeventual plasma cell differentiation. Antibodies can be criticalmolecules in clearance of extracellular pathogens such as variousbacteria and parasites.

Under many circumstances, naive CD4⁺ T cells require two distinctsignals to proliferate and differentiate into the armed effector cellsthat mediate adaptive immunity. Signal 1 of this two-signal model isantigen-specific and is generated by interaction of the TCR withantigenic peptide presented in context with MHC II antigens. Thisresults in transduction of TCR intracellular signals leading toproduction of IL-2 and T cell activation. Signal 2 is referred to as a“costimulatory” signal because, while essential, it does not necessarilyinduce any functional response in T cells.

The best characterized costimulatory signal 2 is generated through the Tcell surface molecule CD28. CD28 delivers a costimulatory signal uponinteraction with CD80 or CD86 present on B cells, macrophages, ordendritic cells. Activation of the TCR in the presence of costimulatorysignals leads to T cell clonal expansion and initiation of effectorfunctions such as IL-2 production.

For cancer, immune inhibitory mechanisms, termed “immune checkpoints,”are prematurely activated in order for the tumor to escape immuneattack. Two immune checkpoints exist: a) CTLA-4, which sends aninhibitory signal to T cells upon binding CD80 or CD86 on antigenpresenting cells; and b) PD-1, which binds to PD-1 ligand on tumorcells, stromal cells, or antigen presenting cells.

CTLA-4 is related to CD28, however instead of activating T cells in aco-stimulatory manner, it leads to inhibition or co-inhibition of Tcells.

Nuclear receptor subfamily 2, group F, member 6 (NR2F6), also known asnuclear orphan receptor Ear2, is an orphan member of the nuclearreceptor (NR) superfamily of ligand-activated receptors, which exhibit acommon modular structure and are involved in various homeostaticfunctions, but also play a role in oncogenesis and cancer propagation.Specifically, studies have shown that members of the NR family regulatedevelopment, reproduction, and metabolism of lipids, drugs and energy.The importance of this family of proteins in metabolic disease isexemplified by NR ligands used in the clinic or under exploratorydevelopment for the treatment of diabetes mellitus, dyslipidemia,hypercholesterolemia, or other metabolic abnormalities.

Genetic studies in humans and rodents support the notion that NRscontrol a wide variety of metabolic processes by regulating theexpression of genes encoding key enzymes, transporters and otherproteins involved in metabolic homeostasis. Genomic sequenceavailability has led to the identification of 48 NRs encoded by thehuman genome and 49 NRs encoded by the mouse genome.

The present disclosure is directed to, in certain embodiments, methodsof using small molecule compounds as immune modulators; as well as tocompounds, solid forms and compositions thereof that are immunemodulators and that exhibit desirable characteristics thereof; as wellas to methods of making the compounds, solid forms and compositionthereof.

SUMMARY OF THE DISCLOSED TECHNOLOGY

In certain embodiments, the present technology is directed compoundsdiscussed and described herein, which compounds have been found tomodulate the immune system. These compounds can include any of thefollowing:

In various embodiments, in any of the above, moieties R, RA, RB, R1-R8,X, Q, Q1, Q2, A can be any of the following: C, H, N, O, S, a halogen,an alkyl group, a substituted alkyl group, a cyclic alkyl group, an arylgroup, a substituted aryl group, a heterocyclic group, an ester, analdehyde, a ketone, a carboxylic acid, an amide, an amine, an ether, athiol or a nitrile. In various embodiments, in any of the above, n canbe an integer 1, 2, 3, 4, 5 or 6.

In various embodiments, any of R, RA, RB, R1-R8, X, Q, Q1, Q2, or A canbe any of the following: Me, OMe, Br, N, H, Cl, F or NO₂. In certainembodiments, any of R, RA, RB, R1-R8, X, Q, Q1, Q2, or A can be any ofthe following: 4-Me, 4-OMe, 4-Br, 4-t-Bu, 3,4-di-Me, 4-Cl, 3,4-di-Cl,3-Cl-4-F, 2-F, 3-Cl, 3-CH₃-4-F, a thiazole, an isothiazole or adithiolane.

In various embodiments, any of R1 and R2 can have the values shown inany of the Figures, for example, FIGS. 1A-1F, FIGS. 3A-3F, FIGS. 4A-4L,FIGS. 5A and 5B, FIGS. 6A-6F, FIGS. 7A-7O, FIGS. 8A-8M, FIGS. 9A-9J,FIGS. 10A-10J or FIGS. 11A-11G.

In certain embodiments, the present technology is directed a compoundhaving a structure of any of the following, or a pharmaceuticallyacceptable salt thereof:

In certain embodiments, the present technology is directed to a novelcompound, any solid form thereof, and any formulation or compositionthereof, that is useful as agonists or antagonists of nuclear receptoractivity, specifically to the modulation of NR2F6 activity and NR2F6utilizing compounds.

In certain embodiments, the present technology is directed to methods ofmodulating the immune system or modulating cancer cell activity usingcompounds that alter activity of NR2F6.

In certain embodiments, the present technology is directed to methods of“reprogramming” the immune cells in a patient to attack tumors or otherinvasive cells. Such “reprogramming” can include: (a) extraction of anamount of a patient's cellular material (including, but not limited to:blood, which itself includes blood serum, plasma red blood cells, whiteblood cells and platelets), (b) isolating specific immune cells from thecellular material; (c) inhibiting or activating the NR2F6 target in theextracted immune cells; and (d) re-administering the immune cells (forexample, by injection) to the patient's body.

In certain embodiments, the present technology is directed to methodstreating or reducing the effect of an autoimmune response, reaction,disease or disorder, the method comprising any of the steps discussedherein, or activating the NR2F6 target in isolated immune cells bybinding them with a compound according to the present technology.

In certain embodiments, the present technology is directed to methods ofshrinking (reducing the size of) a tumor, increasing or decreasing theactivity of a cell, initiating or inducing an immune response,destroying a cancer cell, reducing the effect of a disease, alleviatinga symptom of a disease, treating a disorder, as well as methods ofinducing a cell in a patient's body to do any of these, the methodcomprising administering a compound herein to a tumor, contacting acompound herein with a cell, or any other steps discussed herein. Invarious embodiments, these methods can comprise: comprising the stepsof:

(a) extracting an amount of a patient's cellular material:

(b) isolating immune cells from the cellular material;

(c) activating the NR2F6 target in the isolated immune cells by bindingthem with a compound of claim 1; and

(d) re-administering the isolated immune cells to the patient's body.

In certain embodiments, the present technology is directed to methods oftreating or reducing the effect of a reaction, disease or disorder, themethod comprising activating the NR2F6 target in immune cells bycontacting them with a compound herein.

In certain embodiments, the present technology is directed to apharmaceutical composition comprising a compound described herein, witha pharmaceutically acceptable carrier or excipient.

The methods herein can, in various embodiments, involve humans ornon-human mammals as subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show certain compounds that have been found to be effectivefor the purposes of the present technology.

FIG. 2A shows a certain compound that has been found to be effective forthe purposes of the present technology. FIG. 2B shows different domains,or portions of a base compound, that were substituted with differentmoieties to ascertain whether these made a difference in the activity ofsuch compound.

FIG. 3A shows a certain compound that has been found to be effective forthe purposes of the present technology. FIGS. 3B-3F show variousadditional compounds formed from substitution of different moieties.

FIGS. 4A-4L show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIGS. 5A and 5B show additional embodiments of compounds, along with (inthe case of FIG. 5A) different values of moieties R1 and R2.

FIGS. 6A-6F show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIGS. 7A-7O show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIGS. 8A-8M show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIGS. 9A-9J show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIGS. 10A-10J show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIGS. 11A-11G show additional compounds that were found to be useful inaccordance with the embodiments herein.

FIG. 12 shows a certain compound that has been found to be effective forthe purposes of the present technology: and different domains, orportions of a base compound, that were substituted with differentmoieties to ascertain whether these made a difference in the activity ofsuch compound.

FIG. 13A shows a certain compound that has been found to be effectivefor the purposes of the present technology. FIGS. 13B-13H show variousadditional compounds formed from substitution of different moieties.

FIG. 14A shows a certain compound that has been found to be effectivefor the purposes of the present technology. FIGS. 14B-14D show variousadditional compounds formed from substitution of different moieties.

FIGS. 15A-15G show additional compounds that were found to be useful inaccordance with the embodiments herein

FIGS. 16A and 16B show charts of cytokines release by hPBMC and cytotoxfor Compound C11, which was identified as a useful compound inaccordance with the embodiments herein. Results were repeated in followset screen from fresh powder. 5 direct analogs were available (topstructure in FIG. 16A).

FIGS. 17A and 17B show analogues and other related compounds to CompoundC11.

FIGS. 18A and 18B show charts of cytokines release by hPBMC and cytotoxfor Compound 18. For cytokines release and cytotox on hPBMCs compoundwas tested at 1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. For cytotoxon HEK293, HEK293 pGL4 and HEK293 NR2F6 (full length) compound wastested from 50 uM with dilution step 3.16 in duplicates. Human PBMC wereactivated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized tocontrols with (100%) w/o compounds.

FIGS. 19A and 19B show the results of Dog's PBMC ELISA and cytotoxicityexperiments. All compounds were tested at 5, 10, 25 and 50 uM induplicates on activated by 10 ng/mL PMA+500 ng/mL ionomycin dogs PBMC(1×106 cells/mL). Cell culture supernates were removed and frozen forfurther ELISA analysis and remained cells were analyzed. Compoundswithout cytotox were chosen for cytokine release inhibition analysis.Compound Z92 was also analyzed at 5 uM and 10 uM.

FIGS. 20A and 20B show results of cytokines release by hPBMC and cytotoxfor Compound Z95. For cytokines release and cytotox on hPBMCs compoundwas tested at 1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. For cytotoxon HEK293, HEK293 pGL4 and HEK293 NR2F6 (full length) compound wastested from 50 uM with dilution step 3.16 in duplicates. Human PBMC wereactivated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized tocontrols with (100%) without compounds.

FIGS. 21A-21D show human and dog results of a cytokine releaseexperiment—parent compound for dogs and human PBMC, for Compound D28.All compounds were tested at 5, 10, 25 and 50 uM in duplicates. Dog PBMC(1×106 cells/mL) were activated by 10 ng/mL PMA+500 ng/mL ionomycin.Data were normalized to controls with (100%) without (0%) PMA+ionomycinactivation.

FIGS. 22A and 22B show results of cytokines release by hPBMC and cytotoxfor Compound Z17. For cytokines release and cytotox on hPBMCs compoundwas tested at 1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. For cytotoxon HEK293, HEK293 pGL4 and HEK293 NR2F6 (full length) compound wastested from 50 uM with dilution step 3.16 in duplicates. Human PBMC wereactivated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized tocontrols with (100%) without compounds.

FIGS. 23A and 23B show results of cytokines release by hPBMC and cytotoxfor Compound Z33. For cytokines release and cytotox on hPBMCs compoundwas tested at 1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. For cytotoxon HEK293, HEK293 pGL4 and HEK293 NR2F6 (full length) compound wastested from 50 uM with dilution step 3.16 in duplicates. Human PBMC wereactivated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized tocontrols with (100%) without compounds.

FIG. 24 shows results of a cytokines release by hPBMC for Compound E56.

FIGS. 25A and 25B show results of cytokines release by hPBMC and cytotoxfor Compound Z96. For cytokines release and cytotox on hPBMCs compoundwas tested at 1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. For cytotoxon HEK293, HEK293 pGL4 and HEK293 NR2F6 (full length) compound wastested from 50 uM with dilution step 3.16 in duplicates. Human PBMC wereactivated by 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized tocontrols without (100%) compounds.

FIGS. 26A and 26B show results of cytokine release by hPBMC and cytotoxfor Compound Z97.

FIGS. 27A-27D show NR2F6 and LBD transient transfection, respectively,for Compound D28. FIGS. 27E and 27F show toxicity of Compound D28.

FIGS. 28A-28D show the results of a cytokine release experiment for dogand human PBMC. All compounds were tested at 5, 10, 25 and 50 uM induplicates. Dog PBMC (1×106 cells/mL) were activated by 10 ng/mL PMA+500ng/mL ionomycin. Data were normalized to controls with (100%)/without(0%) PMA+ionomycin activation.

FIGS. 29, 30 31A, 31B and 32 show exemplary methods of formulating thecompounds that have been discussed herein.

FIGS. 33A and 33B and 34A-34D show NR2F6 and LBD transient transfection,respectively, for Compound E21.

FIGS. 35A to 35D show the results of a cytokine release experiment fordog and human PBMC, for Compound E21.

FIG. 36 shows additional compounds related to compound E21 that weretested herein.

FIG. 37 shows additional compounds that were synthesized and testedaccording to the present embodiments.

FIGS. 38A and 38B, 39A-39D and 40A-40D show results of testing onCompound F1.

FIG. 41 shows the general SAR strategy for testing Compound F1 andcompounds related to it in structure; by dividing the active moleculeinto four domains (Domains A through D), and evaluating each domainindependently to establish SAR trends.

FIG. 42 shows an exemplary synthesis of a boronate compound, and theresults of other exemplary syntheses of compounds comprising boronate,and the relative proportions of resultant compounds.

FIG. 43 shows methods of synthesis of certain compounds found to beuseful for the embodiments herein.

FIGS. 44A and 44B and 45A-D show NR2F6 and LBD transient transfection,respectively, for Compound P1.

FIGS. 46A and 46B show the results of a cytokine release experiment fordog and human PBMC, for Compound P1.

FIGS. 47A and 47B show NR2F6 agonist activity of various compoundsdiscussed herein.

FIGS. 48-51 show synthesis of various additional compounds discussedherein.

FIGS. 52A and 52B show synthesis of Compounds Z119, Z120, Z121 and Z123.

FIG. 53-55 shows synthesis of additional compounds herein.

FIGS. 56A and 56B show Nr2F6 agonist activity for various compoundsdiscussed herein.

FIGS. 57 and 58 show synthesis of various compounds discussed herein.

FIGS. 59A-D show further results for firefly, renila and pGL4 forCompounds D104, D118, D122 and D137.

FIGS. 60A-60C show further results for cytokines release inhibition forCompounds D136, D140, D141, D142. Z155 and Z166.

FIGS. 61A-61E show further results of cytokine release by hPBMC forCompound D136.

FIGS. 62A and 62B show stability in plasma (human plasma and rat plasma)and various pH solubility data for Compound D136.

FIGS. 63A and 63B show stability in simulated gastric fluid (SGF) andsimulated intestinal fluid (SIF), respectively, for Compound D136.

FIGS. 64A and 64B show microsomal stability in human liver microsomesand rat liver microsomes, respectively, for Compound D136.

FIGS. 65A and 65B show data for PK in rat plasma for Compound D136.

FIGS. 66A and 66B show data for PK in mice plasma for Compound D136.

DETAILED DESCRIPTION

As used herein. “disease” or “disorder” are used interchangeably andmean a disorder of structure or function in any living thing (includingbut not limited to a human, animal, or plant), especially one thatproduces specific signs or symptoms or that affects a specific locationand is not simply a direct result of physical injury.

As used herein, “mammal” means a warm-blooded vertebrate animal of aclass that is distinguished by the possession of hair or fur, thesecretion of milk by females for the nourishment of the young, and(typically) the birth of live young. As used herein, “human” means aperson. As used herein, “animal” or “non-human mammal” means anynon-human animal, including but not limited to: a canine (e.g., a dog),a feline (e.g., a cat), a rodent, an ungulate (e.g., a cow or ox), anequine (e.g., a horse), or a primate.

As used herein, “modulator” means a molecule that alters the basalactivity of NR2F6 either positively (activates it or increases it) ornegatively (represses, suppresses or decreases it). “Modulating” meansthe act of the modulator, either positive or negative. A compound of thetechnology herein can be, in various embodiments, a modulator of NR2F6,for example, at an effective concentration or in an effective amount,but not be a modulator of any other receptor, or not a modulator at anyother amount of NR2F6 or any other receptor. This can provideselectivity of effect of a compound of the technology herein whenadministered to a patient for treatment of any disease.

In certain embodiments, one diastereomer or one enantiomer of a compoundof the present technology can display superior biological activitycompared with the other. When required, separation of the diastereomericmixture or the racemic material can be achieved by HPLC, optionallyusing a chiral column or by using a resolving agent such as camphonicchloride for the resolution of enantiomers. A chiral compound describedherein can also be directly synthesized using a chiral catalyst or achiral ligand.

In certain embodiments, one deuterated or tritiated compound of thepresent technology can display superior biological activity comparedwith one or more others. When required, separation of the material canbe achieved by one of ordinary skill in the art.

In certain embodiments, the present technology is directed to methods ofmodulating the immune system using compounds that alter activity ofNR2F6.

In certain embodiments, compounds herein can be utilized for stimulationof NR2F6 activity, alone, or in combination with, for example, PKCactivation. In certain embodiments, the compounds herein can be utilizedfor inhibition of NR2F6 activity, alone or in combination with, forexample, anti-PD1, anti-PDL1 or anti-CTLA4 antibodies.

In other embodiments, the methods are directed to the stimulation ofNR2F6 for, e.g., induction of immune inhibition, or stimulation ofcellular proliferation without significant induction of differentiation.Inhibition of NR2F6 can be desirable in situations where a clinicianseeks to augment immune response, or induce cellular differentiation.

In other embodiments, inhibition of NR2F6 expression can be desirable insituations where inhibition of cancer or cancer stem cells is desired.

In certain embodiments, activation of NR2F6 expression can be desirablein situations where inhibition of the immune system is desired, forexample, in connection with autoimmune disorders.

The interplay between the activation or deactivation of NRs by differentstructural classes of endogenous ligands, such as the steroid andthyroid hormones, lipids, vitamins and other biochemicals, is animportant part of their function. The 48 NR family members areclassified into subgroups based on the identification of endogenousligands for each receptor. The endocrine receptors include the steroidhormone receptors that bind steroid hormones and the heterodimericreceptors that partner with the retinoid X receptor and bind thyroidhormones, retinoids, and vitamin D.

The identification of specific endogenous ligands for the endocrinereceptors has facilitated the design and development of selectivereceptor modulators (SRMs) that exhibit tissue-specific agonist orantagonist activities and are used for treatment of hormone-/hormonereceptor-dependent diseases. Tamoxifen is one of many selective estrogenreceptor (ER) modulators used in endocrine therapies for treatingER-positive breast cancer patients.

Adopted orphan receptors are a subtype of NRs that are subdivided intogroups based on their ligands. The lipid sensor receptor subtypes andtheir ligands include retinoid X receptor (9-cis-retinoic acid),peroxisome proliferator-activated receptors (PPARs) (fatty acids), liverX receptor (oxysterols), farnesoid X receptor (bile acids), and pregnaneX receptor, which binds cholesterol derivatives.

Retinoid X receptors have been found in various cancer stem cells andmethodologies for their utilization, as well as ligands/syntheticligands targeting them, have been developed. Any of these can beutilized by one of skill in the art to practice the methods of thepresent technology, which provides compounds useful for modulating theNR2F6 nuclear receptor. Methods of modulating PPARs are also amenable toutilization in the context of the current technology, whosemethodologies can, in various embodiments, be adapted for use with thecompounds discussed herein for treatment of cancer or immune modulation.

With regard to PPARs, three subtypes of the PPAR family are PPARα,PPARγ, and PPARδ, PPARγ, is abundantly expressed in many cell types,where it regulates lipid metabolism, glucose homeostasis, tumorprogression, and inflammation. Polyunsaturated fatty acids, eicosanoids,prostaglandins, and linoleic acid have been identified as endogenousligands for PPARγ. The thiazolidinedione class of compounds function ashigh-affinity synthetic agonists for PPARγ subsequent to exposure tospecific ligands. PPARγ forms a heterodimer complex with retinoid Xreceptor, which then mediates the target gene expression. In terms ofimmune modulation, in certain embodiments, NR2F6 specific compounds canbe substituted for those described for PPAR.

The enigmatic orphan receptor subtype can include the constitutiveandrostane receptor (androstane and many drugs or xenobiotics),hepatocyte nuclear factor-4, and steroidogenic factor-1/liver receptorhomolog 1 (LRH-1) (phospholipids), retinoid acid-related orphan receptor(cholesterol and retinoic acids), and estrogen-related receptor(estrogens). These can be useful in methods of performing immunotherapythat include NR2F6 modulators.

The orphan receptors are the third class of NRs. The crystal structureof the ligand-binding domain of the orphan receptor Nurr1 (NR4A2) showsthat several hydrophobic residues protrude into the ligand-bindingpocket, and a typical coactivator-binding site is lacking, suggestingthat some orphan receptors may not bind ligands.

Like other NR classes, the orphan receptors play important roles incellular homeostasis and diseases including cancer, and several recentreports document the expression and potential functions of orphanreceptors in different tumors and cancer cell lines. Breast tumors areroutinely classified as ER⁺ or ER⁻, and expression of ER has prognosticsignificance that influences selection of therapeutic regimens. However,analysis of ER⁺ and ER⁻ tumors for expression (mRNA) of all 48 NRs alsodemonstrated the important prognostic significance of several orphanreceptors. The NR4A (Nur77/TR3, Nurr1, and Nor1) and NR2F6[v-erbA-related protein (EAR2)] receptors are uniquely overexpressed in(ER⁺ and ER⁻ combined) tumors. Moreover. Nur77, EAR2, and chickenovalbumin upstream promoter transcription factor II (COUP-TFII) areamong a limited group of NRs that are prognostic for breast cancerclassification and histologic grade, and COUP-TFII expression was apositive prognostic factor for tamoxifen-treated ER′ breast cancerpatients.

Examination of lung tumor and nontumor tissue indicated highly variableNR expression; however, gene combinations and individual receptors, suchas the orphan receptor small heterodimer partner (SHP, NR0B2), predictedenhanced survival for early-stage lung cancer patients. Moreover,expression of Nur77 in normal lung epithelium from patients has beenshown to be an indicator for good prognosis.

NR profiling of the NCI60 cancer cell panel demonstrated that relativeexpression levels of some orphan receptors also correlated with drugsensitivity. For example, cancer cell sensitivity tomicrotubule-disrupting drugs has been found to be enhanced in cellsexpressing low levels of NR2F6 and COUP-TFII, whereas high levels of theorphan receptor tailless (TLX, NR2E1) correlated with sensitivity to9-fluoroprednisolone.

As used herein, the term “NR2F6” means “nuclear receptor subfamily 2,group F, member 6” or “Ear2.” Nuclear receptors are transcriptionfactors that regulate the expression of specific target genes, therebyorchestrating a wide array of cellular processes including cellularactivation, development and disease progression. The nuclear receptorsuper-family includes receptors that bind to hormones and orphanreceptors with yet undefined endogenous ligands. As discussed in thepresent disclosure, NR2F6 can be a target in cancer immunotherapy orautoimmune suppression.

The COUP-TF orphan receptors are preferentially expressed in the centralnervous system and organs that depend on the interaction betweenmesenchyme and epithelial layers. The three mammalian COUP-TF familymembers are NR2F1/Ear3, NR2F2/Arp1 and NR2F6. The established targetgenes of said COUP-TF family members are apolipoproteins and retinoicacid-, peroxisome-, oxytocin-, estrogen- and vitamin D receptors. Byyeast 1-hybrid screen and in vitro assays with recombinant NR2F6, it wasfound that the TGACCT direct-repeat motif is the DNA binding sequence ofNR2F6, and that overexpression of NR2F6 induces repression of the reningene transcription in a DNA-binding-specific manner. Wild typehuman/animal NR2F6 is known to possess the following nucleotidesequence: 1 gtgcagcccg tgccccccgc gcgccggggc cgaatgcgcg ccgcgtagggtcccccgggc 61 cgagaggggt gcccggaggg aagagcgcgg tgggggcgcc ccggccccgctgccctgggg 121 ctatggccat ggtgaccggc ggctggggcg gccccggcgg cgacacgaacggcgtggaca 181 aggcgggcgg ctacccgcgc gcggccgagg acgactcggc ctcgccccccggtgccgcca 241 gcgacgccga gccgggcgac gaggagcggc cggggctgca ggtggactgcgtggtgtgcg 301 gggacaagtc gagcggcaag cattacggtg tcttcacctg cgagggctgcaagagctttt 361 tcaagcgaag catccgccgc aacctcagct acacctgccg gtccaaccgtgactgccaga 421 tcgaccagca ccaccggaac cagtgccagt actgccgtct caagaagtgcttccgggtgg 481 gcatgaggaa ggaggcggtg cagcgcggcc gcatcccgca ctcgctgcctggtgccgtgg 541 ccgcctcctc gggcagcccc ccgggctcgg cgctggcggc agtggcgagcggcggagacc 601 tcttcccggg gcagccggtg tccgaactga tcgcgcagct gctgcgcgctgagccctacc 661 ctgcggcggc cggacgcttc ggcgcagggg gcggcgcggc gggcgcggtgctgggcatcg 721 acaacgtgtg cgagctggcg gcgcggctgc tcttcagcac cgtggagtgggcgcgccacg 781 cgcccttctt ccccgagctg ccggtggccg accaggtggc gctgctgcgcctgagctgga 841 gcgagctctt cgtgctgaac gcggcgcagg cggcgctgcc cctgcacacggcgccgctac 901 tggccgccgc cggcctccac gccgcgccta tggccgccga gcgcgccgtggctttcatgg 961 accaggtgcg cgccttccag gaggaggtgg acaagctggg ccgcctgcaggtcgactcgg 1021 ccgagtatgg ctgcctcaag gccatcgcgc tttcacgcc cgacgcctgtggcctctcag 1081 acccggccca cgttgagagc ctgcaggaga aggcgcaggt ggccctcaccgagtatgtgc 1141 gggcgcagta cccgtcccag ccccagcgct tcgggcgcct gctgctgcggctccccgccc 1201 tgcgcgcggt ccctgcctcc ctcatctccc agctgttctt catgcgcctggtggggaaga 1261 cgcccattga gacactgatc agagacatgc tgctgtcggg gagtaccttcaactggccct 1321 acggctcggg ccagtgacca tgacggggcc acgtgtgctg tggccaggcctgcagacaga 1381 cctcaaggga cagggaatgc tgaggcctcg aggggcctcc cggggcccaggactctggct 1441 tctctcctca gacttctatt ttttaaagac tgtgaaatgt ttgtcttttctgttttttaa 1501 atgatcatga aaccaaaaag agactgatca tecaggccte agcctcatcctccccaggac 1561 ccctgtccag gatggagggt ccaatcctag gacagccttg ttcctcagcacccctagcat 1621 gaacttgtgg gatggtgggg ttggcttccc tggcatgatg gacaaaggcctggcgtcggc 1681 cagaggggct gctccagtgg gcaggggtag ctagcgtgtg ccaggcagatcctctggaca 1741 cgtaacctat gtcagacact acatgatgac tcaaggccaa taataaagacatttcctacc 1801 tgca, which corresponds to the following amino acidsequence:

(SEQ ID NO: 1) MAMVTGGWGGPGGDTNGVDKAGGYPRAAEDDSASPPGAASDAEPGDEERPGLQVDCVVCGDKSSGKHYGVFTCEGCKSFFKRSIRRNLSYTCRSNRDCQIDQHHRNQCQYCRLKKCFRVGMRKEAVQRGRIPHSLPGAVAASSGSPPGSALAAVASGGDLFPGQPVSELIAQLLRAEPYPAAAGRFGAGGGAAGAVLGIDNVCELAARLLFSTVEWARHAPFFPELPVADQVALLRLSWSELFVLNAAQAALPLHTAPLLAAAGLHAAPMAAERAVAFMDQVRAFQEQVDKLGRLQVDSAEYGCLKAIALFTPDACGLSDPAHVESLQEKAQVALTEYVRAQYPSQPQRFGRLLLRLPALRAVPASLISQLFFMRLVGKTPIET LIRDMLLSGSTFNWPYGSGQ.

Accordingly, in certain embodiments the present technology is directedto compounds that bind to a portion or all of an NR2F6 molecule; or anymolecule that is, in various embodiments, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to the amino acid sequence of NR2F6.

As used herein, the terms “agonist” or “activator” are usedinterchangeably and mean a compound or substance capable of fully orpartially stimulating the physiologic activity of one or more specificreceptors. In the context of the present disclosure, an agonist cantherefore stimulate the physiological activity of a receptor such asNR2F6 upon binding of said compound substance to said receptor. Asfurther discussed herein, an “agonist” or “activator” can be used to“activate,” “stimulate” or “increase activity” of a cell.

In certain embodiments, binding of an “agonist” or “activator” to agiven receptor, e.g., NR2F6, can mimic the action of an endogenousligand binding to said receptor. As used herein, accordingly, the term“agonist” also encompasses partial agonists or co-agonists orco-activators. In addition, however, an “agonist” or “activator” ofNR2F6 as used herein can also be capable of stimulating the function ofa given receptor, such as NR2F6, by inducing or enhancing the expressionof the nucleic acid molecule encoding for said receptor. Thus, anagonist or activator of NR2F6 can, in certain embodiments, lead to anincreased expression level of NR2F6 (e.g., increased level of NR2F6mRNA, NR2F6 protein) which is reflected in an increased activity ofNR2F6. This increased activity can be measured or detected by themethods herein.

Accordingly, an activator of NR2F6 in accordance with the presenttechnology can, in certain embodiments, also encompass transcriptionalactivators of NR2F6 expression that are capable of enhancing NR2F6function. As mentioned above, “agonist” includes a partial agonist.“Partial agonists” mean candidate molecules that behave like agonists,but that, even at high concentrations, cannot activate NR2F6 to the sameextent as a full agonist. As described below, the increased expressionor activity of NR2F6 by an agonist or activator of NR2F6 can lead todecreased activity (or expression) of components of the NR2F6-dependentsignaling pathway; in particular the activity of NF-AT and AP-1 can bedecreased. NF-AT/AP-1 regulate transcription/expression of further“downstream” components of the NR2F6-dependent signaling pathway, suchas IL-2, IL-17 and/or IFN-gamma. A decrease in NF-AT/AP-1 activity canresult in a decreased transcription of these “downstream” components(e.g., IL-2, IL-17 and/or IFN-gamma) which in turn leads to asuppression of an immune response.

In certain embodiments, an agonist or activator of NR2F6 can lead tosuppression of an immune response. Hence, the use of potentagonists/activators of NR2F6 can lead to a higher expression or activityof NR2F6.

In certain embodiments NR2F6 can be considered its own “agonist” or“activator.” For example, in certain embodiments, overexpression ofNR2F6 can lead to enhanced NR2F6 activity, thus agonizing NR2F6function. Accordingly, NR2F6 as defined herein can, in certainembodiments, be used for the treatment of a disease related to anaugmented immune response.

For example, NR2F6 can be used in accordance with certain embodiments ofthe present technology, wherein NR2F6 is any of the following: (a) apolypeptide comprising an amino acid encoded by a nucleic acid moleculehaving the nucleic acid sequence of NR2F6: (b) a polypeptide having anamino acid sequence of NR2F6; (c) a polypeptide encoded by a nucleicacid molecule encoding a peptide having an NR2F6 amino acid sequence;(d) a polypeptide comprising an amino acid encoded by a nucleic acidmolecule hybridizing to the complementary strand of nucleic acidmolecules as defined in (a) or (c) and encoding a NR2F6 or a functionalfragment thereof: (c) a polypeptide having at least 60% homology to thepolypeptide of any one of (a) to (d), whereby said polypeptide is aNR2F6 or a functional fragment thereof: or (f) a polypeptide comprisingan amino acid encoded by a nucleic acid molecule being degenerate as aresult of the genetic code to the nucleotide sequence of a nucleic acidmolecule as defined in (a), (c) and (d). As described herein, theincrease of NR2F6 activity can lead to a decreased activity ofNF-AT/AP-1 (and other components of the NR2F6-dependent signalingpathway) which in turn can result in a suppressed immune response.

An exemplary transfection of CD4⁺ T cells with a construct for theoverexpression of NR2F6 is also shown in the appended examples. Asdemonstrated therein, overexpression (about 5-fold increase over normalexpression level) can lead to a diminished IL-2 activity/expression andconsequently to a reduced IL-2 amount, resulting in a reduced immuneresponse.

Therefore, agonists/activators of NR2F6 are useful in the treatment ofdiseases where suppression of the immune response is desired (e.g.,diseases with an overstimulated immune response, such as allergies andmultiple sclerosis). As used herein, the term “overexpression” meansthat the NR2F6 activity/expression is, in various embodiments, at least1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least9-fold, at least 10-fold, or at least 25-fold increased in comparison toa (control) standard value as defined herein, wherein a value of 25 foldexpression level or greater over normal can be considered as a maximumoverexpression level.

As used herein, “antagonist” or “inhibitor” are used interchangeably andmean a compound or substance capable of fully or partially suppressingor inhibiting the physiologic activity of one or more specificreceptors. In the context of the present disclosure, an antagonist cantherefore suppress the physiological activity of a receptor upon bindingof said compound substance to said receptor but does not activate thereceptor and therefore blocks the activity of other agonists. As furtherdiscussed herein, an “antagonist” or “inhibitor” can be used to“deactivate,” “inhibit,” “suppress” or “decrease activity” of a cell.

As used herein, the terms “immune response” or “immune reaction” areused interchangeably and mean the response or reaction of the immunesystem to an antigen. In the case of an immune response, immune cellsare activated in such way that one or more specific functions of saidimmune cells can be induced. The “immune cells” can include, but are notlimited to, B cells, T cells, neutrophils, eosinophils, basophils, mastcells, macrophages and dendritic cells. In certain embodiments, said“specific function(s) of activated immune cells” can include, but arenot limited to, secretion of antibodies, presentation of antigen,proliferation of said immune cells, secretion of cytokines such asinterleukin-2 (IL-2), interleukin-17 (IL-17), interleukin-18 (IL-18), orinterferon gamma (IFNgamma), expression of regulatory-, activation- oradhesion molecules, and the ability to induce apoptosis or cytolysis.

As used herein, the term “antigen” means any substance capable ofinducing an immune response. An antigen typically is associated with aforeign substance (i.e. a “non-self antigen”). However, an ownbody-derived substance (i.e., a “self antigen”) can also induce animmune response.

As used herein, accordingly, the term “immune response” also encompassesautoimmune responses or autoimmune reactions. For example, in certainembodiments, the technology herein is directed to a method of treatingor reducing the effect of an autoimmune response, reaction, disease ordisorder, the method comprising activating the NR2F6 target in isolatedimmune cells by binding them with a compound according to the presenttechnology.

As used herein. “treating a cancer,” “inhibiting cancer” or “reducingcancer growth” are used interchangeably and mean inhibiting orpreventing oncogenic activity of cancer cells. Oncogenic activity cancomprise stimulating migration, invasion, drug resistance, cellsurvival, anchorage-independent growth, non-responsiveness to cell deathsignals, angiogenesis, or combinations thereof of the cancer cells. Invarious embodiments, agents suitable for use in treating a cancer orreducing the growth rate of a tumor include, but are not limited to,small organic molecules, peptides, proteins, peptidomimetics, nucleicacids, antibodies and combinations thereof. In various embodiments, suchagents can be formulated with a pharmaceutically acceptable carrier, andcan be administered: intravenously, orally, buccally, sublingually,parenterally, by inhalation, by nasal administration, by insufflation,by topical application, transdermally, by cutaneous injection, or bylocal administration. An agent can additionally be administered inconjunction with one or more anti-cancer chemotherapeutic agent in anadditive or synergistic manner.

As used herein, “cancer,” “cancer cell.” “tumor” and “tumor cell” areused interchangeably and mean a group of diseases characterized byuncontrolled, abnormal growth of cells (e.g., a neoplasia). These caninclude solid tumor cancer, liquid tumor cancer and metastatic disease.In some forms of cancer, the cancer cells can spread locally or throughthe bloodstream and lymphatic system to other parts of the body(“metastatic cancer”). As used herein, “ex vivo activated lymphocytes,”“lymphocytes with enhanced antitumor activity” and “dendritic cellcytokine induced killers” are used interchangeably and mean compositionof cells that have been activated ex vivo and subsequently reintroducedwithin the context of the current disclosure. Although the word“lymphocyte” is used, this also includes heterogeneous cells that havebeen expanded during the ex vivo culturing process including dendriticcells, NKT cells, gamma delta T cells, and various other innate andadaptive immune cells.

As used herein, “cancer” means any disease caused by uncontrolleddivision or growth of abnormal cells, and any malignant growth or tumorresultant from such uncontrolled division or growth. As used herein,“cancer” includes all types of cancer or neoplasm or malignant tumorsfound in animals, including leukemias, carcinomas and sarcomas. Examplesof cancers include, but are not limited to: cancer of the brain, skin(including melanoma), breast, cervix, head and neck, kidney, lung,non-small cell lung, mesothelioma, sarcoma, any internal organ(including bladder, stomach, liver, pancreas, uterus, ovary, prostate,colon) and Medulloblastoma.

As used herein, “leukemia” means a broadly progressive, malignantdisease of the hematopoietic organs or systems and is generallycharacterized by a distorted proliferation and development of leukocytesand their precursors in the blood and bone marrow. Leukemia diseasesinclude, but are not limited to: acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, acute promyelocytic leukemia, adult T-cell leukemia, B celllymphoma, aleukemic leukemia, a leukocythemic leukemia, basophilicleukemia, blast cell leukemia, bovine leukemia, chronic myelocyticleukemia, leukemia cutis, embrvonal leukemia, eosinophilic leukemia.Gross' leukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, undifferentiated cell leukemia,hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia,histiocytic leukemia, stem cell leukemia, acute monocytic leukemia,leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia,lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia,lymphosarcoma cell leukemia, mast cell leukemia, megakaryocvticleukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblasticleukemia, myelocytic leukemia, chronic myeloid leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, and promyelocytic leukemia.

As used herein, the term “carcinoma” means a malignant new growth madeup of epithelial cells tending to infiltrate the surrounding tissues, orresist physiological and non-physiological cell death signals and giverise to metastases. Exemplary carcinomas include, but are not limitedto: acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoidcystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex,alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma,carcinoma basocellulare, basaloid carcinoma, basosquamous cellcarcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma,epiennoid carcinoma, carcinoma epitheliale adenoides, exophyticcarcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniformcarcinoma, gelatinous carcinoma, giant cell carcinoma, signet-ring cellcarcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma,spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum,squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrmcous carcinoma, carcinomavillosum, carcinoma gigantocellulare, glandular carcinoma, granulosacell carcinoma, hair-matrix carcinoma, hematoid carcinoma,hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma,hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma insitu, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, and carcinoma scroti.

As used herein, the term, “sarcoma” means a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar, heterogeneous, orhomogeneous substance. Sarcomas include, but are not limited to:chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma,osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma,fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, granulocyticsarcoma. Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagicsarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblasticsarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cellsarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma,parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocysticsarcoma, synovial sarcoma, and telangiectaltic sarcoma. Additionalexemplary neoplasias include, for example, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer.

In some particular embodiments of the present technology, the cancertreated is a melanoma. As used herein, the term “melanoma” means a tumorarising from the melanocytic system of the skin or other organs.Melanomas include, for example, Harding-Passey melanoma, juvenilemelanoma, lentigo maligna melanoma, malignant melanoma,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, nodular melanoma subungualmelanoma, and superficial spreading melanoma. As used herein, the term“polypeptide” is used interchangeably with “peptide,” “altered peptideligand” and “fluorocarbonated peptides.”

As used herein, the term “pharmaceutically acceptable carrier” means anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. Except insofar as any conventional media or agent is incompatiblewith the active compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions herein.

As used herein, the terms “T cell” or “T lymphocyte” are usedinterchangeably, and mean a cell derived from thymus among lymphocytesinvolved in an immune response. In various embodiments a T cell includesany of: a CD8-positive T cell (cytotoxic T cell: CTL), a CD4⁺ T cell(helper T cell), a suppressor T cell, a regulatory T cell such as acontrolling T cell, an effector cell, a naive T cell, a memory T cell,an alpha (α) beta (β) T cell expressing TCR α and β chains, and a gamma(γ) delta (δ) T cell expressing TCR γ and δ chains.

In certain embodiments, the T cell includes a precursor cell of a T cellin which differentiation into a T cell is directed. Examples of “cellpopulations containing T cells” include, in addition to body fluids suchas blood (peripheral blood, umbilical blood etc.) and bone marrowfluids, cell populations containing peripheral blood mononuclear cells(PBMC), hematopoietic cells, hematopoietic stem cells, umbilical bloodmononuclear cells etc., which have been collected, isolated, purified orinduced from the body fluids.

Further, a variety of cell populations containing T cells and derivedfrom hematopoietic cells can be used in connection with the embodimentsof the present technology. These cells may have been activated bycytokine such as IL-2 in vivo or ex vivo, and can be collected in anyknown way, for example, collected from a living body; obtained via exvivo culture, for example, a T cell population obtained by a methodherein; or obtained by freeze preservation.

As used herein, the term “antibody” means both intact molecules as wellas fragments thereof that include the antigen-binding site. Wholeantibody structure is often given as H₂L₂ and refers to the fact thatantibodies commonly comprise 2 light (L) amino acid chains and 2 heavy(H) amino acid chains. Both chains have regions capable of interactingwith a structurally complementary antigenic target. The regionsinteracting with the target are referred to as “variable” or “V” regionsand are characterized by differences in amino acid sequence fromantibodies of different antigenic specificity. The variable regions ofeither H or L chains contain the amino acid sequences capable ofspecifically binding to antigenic targets. Within these sequences aresmaller sequences dubbed “hypervariable” because of their extremevariability between antibodies of differing specificity. Suchhypervariable regions are also referred to as “complementaritydetermining regions” or “CDR” regions. These CDR regions account for thebasic specificity of the antibody for a particular antigenic determinantstructure. The CDRs represent non-contiguous stretches of amino acidswithin the variable regions but, regardless of species, the positionallocations of these critical amino acid sequences within the variableheavy and light chain regions have been found to have similar locationswithin the amino acid sequences of the variable chains. The variableheavy and light chains of all antibodies each have 3 CDR regions, eachnon-contiguous with the others (termed L1, L2, L3, H1, H2, H3) for therespective light (L) and heavy (H) chains.

In various embodiments, the antibodies discussed herein can also bewholly synthetic, wherein the polypeptide chains of the antibodies aresynthesized and, possibly, optimized for binding to the polypeptidesdisclosed herein as being receptors. Such antibodies can be, in variousembodiments, chimeric or humanized antibodies, and can be fullytetrameric in structure, or can be dimeric and comprise only a singleheavy and a single light chain.

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” are used interchangeably and mean a dosage sufficientto treat, inhibit, or alleviate one or more symptoms of a disease statebeing treated or to otherwise provide a desired pharmacologic orphysiologic effect, especially enhancing T cell response to a selectedantigen. The precise dosage in any given embodiment can vary accordingto a variety of factors such as subject-dependent variables (e.g., age,immune system health, etc.), the disease, and the treatment beingadministered.

As used herein, the terms “individual,” “host,” “subject” and “patient”are used interchangeably and mean a mammal, including, but not limitedto, primates, for example, human beings, as well as rodents, such asmice and rats, and other laboratory animals or any other animalsmentioned herein. In various embodiments, the compounds or compositionsdiscussed herein can be general compounds or compositions useful for anypurpose, pharmaceutical compounds or compositions, or animal (e.g.,veterinary) compounds or compositions.

As used herein, “treat,” “treating” or “treatment” means an alleviationof symptoms associated with a disorder or disease, or inhibition offurther progression or worsening of those symptoms, or prevention orprophylaxis of the disease or disorder; and includes: (i) preventing apathologic condition from occurring (e.g., prophylaxis); (ii) inhibitingthe pathologic condition or arresting its development (e.g., slowing orstopping proliferation of cancer cells or tumor growth): (iii) relievingthe pathologic condition; or (iv) diminishing symptoms associated withthe pathologic condition.

As used herein, the term “treatment regimen” means a treatment of adisease or a method for achieving a desired physiological change, suchas increased or decreased response of the immune system to an antigen orimmunogen, such as an increase or decrease in the number or activity ofone or more cells, or cell types, that are involved in such response. Invarious embodiments discussed herein, the treatment or method comprisesadministering to an animal, such as a mammal, a sufficient amount of oneor more (in certain embodiments two or more) chemical agents orcomponents of said regimen to effectively treat a disease or to producesaid physiological change. In certain embodiments, the two or moreagents or components are administered together, such as part of the samecomposition, or administered separately and independently at the sametime or at different times (i.e., administration of each agent orcomponent is separated by a finite period of time from one or more ofthe agents or components). In certain embodiments, administration ofsaid one or more agents or components achieves a result greater thanthat of any of said agents or components when administered alone or inisolation.

As used herein, the terms “anergy” or “unresponsiveness” are usedinterchangeably and include unresponsiveness to an immune cell tostimulation, for example, stimulation by an activation receptor orcytokine. Anergy can occur due to, for example, exposure to an immunesuppressor or exposure to an antigen in a high dose. Such anergy isgenerally antigen-specific, and can continue even after completion ofexposure to a tolerized antigen. For example, the anergy in a T celland/or NK cell can be characterized by failure of production ofcytokine, e.g., interleukin (IL)-2. The T cell anergy and/or NK cellanergy can occur in part when a first signal (signal via TCR or CD-3) isreceived in the absence of a second signal (costimulatory signal) uponexposure of a T cell and/or NK cell to an antigen.

As used herein, the terms “enhanced function of a T cell,” “enhancedcytotoxicity” and “augmented activity” are used interchangeably and meanthat the effector function of the T cell or NK cell is improved. Incertain embodiments, the enhanced function of the T cell or NK cell caninclude any of the following: an improvement in the proliferation rateof the T cell or NK cell, an increase in the production amount ofcytokine, or an improvement in cytotoxity. Further, in certainembodiments the enhanced function of the T cell or NK cell includescancellation or suppression of tolerance of the T cell or NK cell in thesuppressed state such as the anergy (unresponsive) state, or the reststate, that is, transfer of the T cell or NK cell from the suppressedstate into the state where the T cell or NK cell responds to stimulationfrom the outside.

As used herein, “expression” means generation of mRNA by transcriptionfrom nucleic acids such as genes, polynucleotides, and oligonucleotides,or generation of a protein or a polypeptide by transcription from mRNA.Expression can be detected by any method including RT-PCR, NorthernBlot, or in situ hybridization. As used herein, “suppression ofexpression” means a decrease of a transcription product or a translationproduct in a significant amount as compared with the case of nosuppression. The suppression of expression herein shows, in variousembodiments, a decrease of a transcription product or a translationproduct in amounts of 30% or more, 50% or more, 70% or more, or 90% ormore.

As used herein, “augmented immune response” means characterized by aparticularly strong response or reaction of the immune system to thepresence of an antigen. Under normal, non-pathological conditions,immune responses are regulated in a tightly controlled fashion.Moreover, immune responses are self-limiting and decline in time afterexposure to the antigen. In case of an “augmented immune response”however, the immune response can be hypersensitive, i.e., the immuneresponse can cause damage to the organism's own cells or tissue inpresence of an antigen. Furthermore, in some cases of an “augmentedimmune response,” for example in auto-immune diseases or disorders or intransplant rejects (and the like), the immune system can fail todistinguish between self and non-self substances. As used herein,“disease related to an augmented immune response” accordingly relates toany disease or disorder in which an augmented immune response isetiological for, associated with, secondary to or the resultant of saiddisorder.

In certain embodiments, an augmented immune response can be determinedby directly or indirectly measuring parameters that are indicative forthe magnitude of the immune response or reaction to an antigen, andcomparing the outcome of said measurement with the outcome of the sametest in a physiologically normal subject. Parameters indicative for themagnitude of the immune response/reaction can include, but are notlimited to: the presence or quantity of (specific) antibodies; thepresence or quantity of (specific) immune cells; the presence orquantity of (specific) cytokines: or the presence or quantity of(specific) regulatory-, activation- or adhesion molecules.

For a disease to be related to an augmented immune response,accordingly, said augmented immune response can be detectable preceding,during or following said disease. In certain embodiments, the diseaserelated to an augmented immune response is any of the following:

-   -   an acute or chronic transplant rejection, including septic        shock, infections caused by bacteria including MRSA and viruses;    -   a dermatological disease, for example, psoriasis, atopic        dermatitis or contact allergy;    -   T- and B-cell-mediated inflammatory disease, for example, asthma        or chronic obstructive pulmonary disease (COPD);    -   graft-versus-host disease, for example, acute (or fulminant)        graft-versus-host disease or chronic graft-versus-host disease;        or    -   auto-immune disease, for example, multiple sclerosis,        inflammatory bowel disease, like ulcerative colitis or Behcet's        disease; vasculitis, lupus erythematosus, pemphigus vulgaris,        pemphigus foliaceus, myasthenia gravis, polymyositis, mixed        collective tissue disease (MCTD) rheumatoid arthritis, diabetes        mellitus (whether Type 1 or Type 2), celiac disease, celiac        sprue disease, atherosclerosis, Goodpasture's syndrome, Grave's        disease, autoimmune hepatitis/hepatic autoimmune diseases,        autoimmune thrombocytopenic purpura, granulomatosis (e.g.,        morbus Wegener), Sjogren's Syndrome, scleroderma, alopecia        areata or autoimmune hemolytic anemia.

Immune responses can be exquisitely controlled, requiring multiplefinely tuned levels of activation as well as inactivation signals. In Tlymphocytes among these signaling networks, T cell receptor (TCR)stimulation activates NF-AT/AP-1, a family of transcription factors thatis of particular importance during immune cell activation. NF-ATmediates the transcriptional induction of “cell fate-determining genes,”which govern as diverse outcomes as activation, anergy or apoptosis.Mechanistically, the rise of intracellular Ca²⁺ triggered by antigenbinding to the TCR can lead to the activation of calcincurin'sphosphatase activity. This leads to dephosphorylation of phospho-siteswithin the N-terminal regulatory domain on NF-AT and, subsequently,nuclear import of NF-AT. Upon transient stimuli, however, feedbackinhibition, mediated via GSK3 (glycogen synthase kinase 3), CK 1 (caseinkinase 1) and DYRK (dual-specificity tyrosine phosphorylation-regulatedkinase) protein kinases can counter-regulate NF-AT nuclear occupancy byrephosphorylation, which induces the nuclear export of NF-AT and theabort of immune activation-associated gene transcription. NF-AT familymembers are also subject to regulation in the nucleus through theirability to directly interact with other transcriptional regulatoryfactors. NF-AT requires a protein partner for high-affinity binding atmost DNA sites. NF-AT complexes mostly contain cell type- or celllineage-specific protein binding partners. In cardiac, skeletal, andsmooth muscle cells, NF-AT forms complexes with GATA proteins.

Accordingly, in certain embodiments the present technology is directedto agonists or activators of NR2F6 for the treatment of a diseaserelated to an augmented immune response. In other embodiments, thepresent technology is directed to the use of an agonist or activator ofNR2F6 for the preparation of a medicament for the treatment of a diseaserelated to an augmented immune response. The utilization of NR2F6modulating compounds for alteration of immune response can be utilizedby administering in patients suffering from cancer in which increasedefficacy of a cancer vaccine is desired. In these situations, inhibitionof NR2F6 is desirable, optionally in addition to immune stimulation.Thus, in various embodiments, the compositions herein can comprise anyof the following:

(a) agonists/activators of NR2F6;

(b) antagonists/inhibitors of NR2F6;

(c) agonists/activators of NR2F6 in combination with: (i) one or moreadditional immune enhancers (ii) CAR-T cell therapy (which can reduceside effects); or (iii) autologous cell therapies, e.g., dendriticcells, anti-PD1 and antiCTLA4 antibodies, PMBC, or umbilical vein cordblood-derived cells.

(d) antagonists/inhibitors of NR2F6 in combination with: (i) one or moreadditional immune suppressants; (ii) CAR-T cell therapy (which canreduce side effects); or (iii) autologous cell therapies, e.g.,dendritic cells, anti-PD1 and antiCTLA4 antibodies, PMBC, or umbilicalvein cord blood-derived cells.

Accordingly, in certain embodiments, inhibitor compounds of NR2F6 areadministered with a cancer antigen, said cancer antigens include ROBO-4.In certain embodiments, the antigens can be used to replace ROBO-4.These can include any of the following: a) Fos-related antigen 1: b)LCK; c) FAP; d) VEGFR2: e) NAl7: f) PDGFR-beta: g) PAP; h) MAD-CT-2; i)Tie-2; j) PSA; k) protamine 2: l) legumain; m) endosialin: n) prostatestem cell antigen; o)carbonic anhydrase IX: p) STn: q) Page4: r)proteinase 3; s) GM3 ganglioside; t) tyrosinase; u) MARTI: v) gp100; w)SART3; x) RGS5; y) SSX2: z) Globoll: aa) Tn; ab) CEA: ac) hCG; ad)PRAME; ae) XAGE-1; af) AKAP-4: ag) TRP-2: ah) B7H3; ai) sperm fibroussheath protein; aj) CYPIBI: ak) HMWMAA: al) sLe(a); am) MAGE A1: an)GD2: ao) PSMA; ap) mesothelin: aq) fucosyl GMI; ar) GD3; as) spermprotein 17: at) NY-ESO-1; au) PAX5; av) AFP; aw) polysialic acid; ax)EpCAM; ay) MAGE-A3; az) mutant p53; ba) ras: bb) mutant ras: be) NY-BR1;bd) PAX3: be) HER2/ncu; bf) OY-TESI: bg) HPV E6 E7: bh) PLAC1 bi) hTERT:bj) BORIS; bk) ML-IAP; bl) idiotype of b cell lymphoma or multiplemyeloma: bm) EphA2: bn) EGFRvIII; bo) cyclin B1; bp) RhoC; bq) androgenreceptor; br) surviving; bs) MYCN; bt) wildtype p53: bu) LMP2; by)ETV6-AML: bw) MUC1; bx) BCR-ABL: by) ALK: bz) WTI; ca) ERG (TMPRSS2 ETSfusion gene): cb) sarcoma translocation breakpoint; cc) STEAP; cd)OFA/iLRP; and ce) Chondroitin sulfate proteoglycan 4 (CSPG4).

In certain embodiments, the assessment of compounds for NR2F6 modulatingactivity is performed utilizing means known in the art, such asdescribed in U.S. Pat. No. 9,091,696. Compounds useful for the screeningand modification for enhanced NR2F6 modulatory activity include: CARAgonists such as 5β-Dihydroprogesterone, 6,7-Dimethylesculetin,Amiodarone, Artemisinin, Benfuracarb, Carbamazepine, Carvedilol,Chlorpromazine, Chrysin, CITCO, Clotrimazole. Cyclophosphamide,Cypermethrin, DHEA, Efavirenz, Ellagic acid, Griseofulvin, Methoxychlor,Mifepristone, Nefazodone, Nevirapine, Nicardipine, Octicizer,Permethrin, Phenobarbital, Phenytoin, Reserpine, TCPOBOP, Telmisartan,Tolnaftate, Troglitazone, Valproic acid. CAR Antagonists such as3,17β-Estradiol, 3α-Androstanol, 3α-Androstenol, 3β-Androstanol,17-Androstanol, AITC, Ethinyl estradiol, Meclizine, Nigramide J, Okadaicacid, PK-11195, S-07662, T-0901317. FXR Agonists such as Bile acids,Cafestol, Chenodeoxycholic acid, Fexaramine, GW-4064, Obeticholic acid.FXR Antagonists such as Guggulsterone. LXR Agonists such as22R-Hydroxycholesterol, 24S-Hydroxycholesterol, 27-Hydroxycholesterol,Cholestenoic acid, DMHCA, GW-3965, Hypocholamide, T-0901317, PPAR-alphaAgonists such as 15-HETE, 15-HpETE, Aleglitazar, Aluminum clofibrate,Arachidonic acid, Bezafibrate, Clofibrate, CP-775146, DHEA, Elafibranor,Fenofibrate. Gemfibrozil, GW-7647, Leukotriene B4, LG-101506, LG-100754,Lobeglitazone, Muraglitazar, Oleylethanolamide, Palmitoylethanolamide,Pemafibrate, Perfluorononanoic acid, Perfluorooctanoic acid,Pioglitazone, Saroglitazar, Sodelglitazar, Tesaglitazar,Tetradecylthioacetic acid, Troglitazone. WY-14643. PPAR-alphaAntagonists such as GW-6471. MK-886. PPAR-delta Agonists such as15-HETE, 15-HpETE, Arachidonic acid, Bezafibrate, Elafibranor, GW-0742,GW-501516, L-165,041, LG-101506, MBX-8025, Sodelglitazar,Tetradecylthioacetic acid. PPAR-delta Antagonists such as FH-535,GSK-0660, GSK-3787. PPAR gamma agonists such as 5-Oxo-ETE,5-Oxo-15-hydroxy-ETE, 15-Deoxy-A12,14-prostaglandin J2, 15-HETE,15-HpETE, Aleglitazar, Arachidonic acid, Berberine, Bezafibrate,Ciglitazone, Darglitazone, Edaglitazone, Etalocib, GW-1929, Ibuprofen,LG-100268. LG-100754, LG-101506, Lobeglitazone, Muraglitazar, nTZDpa,Perfluorononanoic, acid, Pioglitazone, Prostaglandin J2, Rosiglitazone,RS5444, Saroglitazar, Sodelglitazar, Telmisartan, Tesaglitazar,Troglitazone. SSPARMS such as BADGE, EPI-001, INT-131, MK-0533, S26948.PPAR gamma antagonists such as FH-535, GW-9662, SR-202, T-0070907. PPARnonselective agonists such as Ciprofibrate, Clinofibrate, Clofibride,Englitazone, Etofibrate, Farglitazar, Netoglitazone, Ronifibrate,Rivoglitazone, Simfibrate. PXR Agonists such as 5α-Dihydroprogesterone,5β-Dihydroprogesterone, 17α-Hydroxypregnenolone,17α-Hydroxyprogesterone. Δ4-Androstenedione, Δ5-Androstenediol,Δ5-Androstenedione, AA-861, Allopregnanolone, Alpha-Lipoic acid,Ambrisentan, AMI-193, Amlodipine besylate, Antimycotics, Artemisinin,Aurothioglucose, Bile acids, Bithionol, Bosentan, Bumecaine, Cafestol,Cephaloridine, Cephradine, Chlorpromazine, Ciglitazone, Clindamycin,Clofenvinfos, Chloroxine, Clotrimazole, Colforsin, Corticosterone,Cyclophosphamide, Cyproterone acetate, Demecolcine, Dexamethasone, DHEA,DHEA-S, Dibunate sodium, Diclazuril, Dicloxacillin, Dimercaprol,Dinaline, Docetaxel, Docusate calcium, Dodecylbenzenesulfonic acid,Dronabinol, Droxidopa. Ebumamoninc, Ecopipam, Enzacamene, Epothilone B.Erythromycin, Famprofazone, Febantel, Felodipine, Fenbendazole,Fentanyl. Flucloxacillin, Fluorometholone, Griseofulvin, Haloprogin,Hetacillin potassium, Hyperforin (Hypericum perforatum), Indinavirsulfate, Lasalocid sodium, Levothyroxine, Linolenic acid, LOE-908,Loratadine, Lovastatin, Meclizine. Methacycline. Methylprednisolone,Metyrapone, Mevastatin, Mifepristone, Nafcillin, Nicardipine, Nicotine,Nifedipine, Nilvadipine, Nisoldipine, Norelgestromin, Omeprazole,Orlistat, Oxatomide, Paclitaxel, Phenobarbital. Plicamycin,Prednisolone, Pregnanolone, Pregnenolone. Pregnenolone 16α-carbonitrile,Proadifen, Progesterone, Reserpine, Reverse triiodothyronine Rifampicin,Rifaximin, Rimexolone, Riodipine, Ritonavir, Simvastatin, Sirolimus,Spironolactone, Spiroxatrine, SR-12813, Suberoylanilide, Sulfisoxazole,Suramin, Tacrolimus, Tenylidone, Terconazole, Testosterone isocaproate,Tetracycline, Thiamylal sodium, Thiothixene, Thonzonium bromide,Tianeptine, Troglitazone, Troleandomycin, Tropanyl 3,5-dimethulbenzoate,Zafirlukast, Zearalanol. PXR Antagonist such as Ketoconazole. RARAgonists such as 9CDHRA, 9-cis-Retinoic acid (alitretinoin), AC-261066,AC-55649, Acitretin, Adapalene, all-trans-Retinoic acid (tretinoin),AM-580, BMS-493, BMS-753, BMS-961, CD-1530, CD-2314, CD-437, Ch-55, EC23, Etretinate, Fenretinide, Isotretinoin, Palovarotene, Retinoic acid.Retinol (vitamin A), Tamibarotene, Tazarotene. Tazarotenic acid, TTNPB.RAR Antagonists such as BMS-195614, BMS-493, CD-2665, ER-50891, LE-135,MM-11253. RXR Agonists such as 9CDHRA, 9-cis-Retinoic acid(alitretinoin), all-trans-Retinoic acid (tretinoin), Bexarotene, CD3254, Docosahexaenoic acid, Fluorobexarotene, Isotretinoin. LG-100268,LG-101506, LG-100754, Retinoic acid, Retinol (vitamin A), SR-11237. RXRAntagonists such as HX-531, HX-630, LG-100754, PA-452, UVI-3003, TRAgonists such as Dextrothyroxine, GC-1, Levothyroxine, Liothyronine,Thyroxine, Tiratricol, Triiodothyronine.

Other useful for modulation of NR2F6 activity include:5-tert-butyl-N-[(6-fluoro-4H-1,3-benzodioxin-8-yl)methyl]-2-methylpvrazole-3-carboxamide,ST50775950, ethyl 4-(cyclohexylamino)-2-(3,5-dimethylpyrazol-1-yl)pynimidine-5-carboxylate, ethyl4-(cyclopentylamino)-2-(3,5-dimethylpyrazol-1-yl)pyrimidine-5-carboxylate,AGN-PC-09SAX3, SMR000064686, AGN-PC-ONLTEQ, T6090485, MLS002548992,5,6-imethyl-4-[4-[2-(4-methylphenoxy)ethyl]piperazin-1-yl]thieno[2,3-d]pyrimidine,MLS002473459, MLS001030349,4-(3,4-dihydro-1H-isoquinolin-2-yl)-5H-pyrimido[5,4-b]indole,4-(3,4-Dihydro-1H-isoquinolin-2-yl)-8-fluoro-5H-pyrimido[5,4-b]indole,4-[4-(4-methoxyphenyl)piperazino]-5H-pyrimido[5,4-b]indole,4-[4-(1,3-benzodioxol-5-ylmethyl)piperazin-1-yl]-7-methoxy-5H-pyrimido[5,4-b]indole,SMR000044829,8-fluoro-N-(3-propan-2-yloxypropyl)-5H-pyrimido[5,4-b]indol-4-amine,GNF-Pf-1678, MLS003116118,2-[4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-1-yl]-1,3-benzothiazole,5-methyl-3,6-diphenylpyrazolo[1,5-a]pyrimidin-7-amine,4-[4-[(4-chlorophenyl)methyl]piperazin-1-yl]-1-[(4-methylphenyl)methyl]pyrazolo[3,4-d]pyrimidine,MLS002632722, MLS002477203, MLS003120814, AGN-PC-07AHX3, MLS003120821,MLS003120807, MLS003120811, MLS003120820, ethyl4-[[1-(2,4-dimethylphenyl)pyrazolo[3,4-d]pyrimidin-4-yl]amino]piperidine-1-carboxylate,N-[2-(3,4-dimethoxyphenyl)ethyl]thieno[2,3-d]pyrimidin-4-amine,N-[2-(3,4-dimethoxyphenyl)ethyl]-6-methylthieno[2,3-d]pyrimidin-4-aminehydrochloride, N-(1-phenylethyl)quinazolin-4-amine, AG-F-87638,ZINC03428816, CHEMBL493153, ST50323391, N-Benzylquinazolin-4-amine,ST50483228,N-[4-(2-methyl-1-methylsulfonyl-2,3-dihydroindol-5-yl)-1,3-thiazol-2-yl]-2-thiophen-2-ylacetamide,F0558-0175, AC1MLRO7,4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-(3-methylphenyl)-1,3-thiazol-2-amine,AGN-PC-09PPXW, Compound 15Jf, AC1MEEXM, ST5094 1838,[2-[(3-carbamoylthiophen-2-yl)amino]-2-oxoethyl]2-naphthalen-1-ylacetate, F0239-0029, AC10BZ00, ST4126227,1-[(4-bromophenyl)methyl]-2-methylbenzimidazole, SMR000718391,MLS002694437, Chlormidazole,2-methyl-1-(2-methylbenzyl)-1H-benzimidazole, MLS003119103,Ambcb90456311, AGN-PC-4RX4B, MLS001122505, Ambcb81049924, AGN-PC-04RX7E,Ambcb42757923, MLS001124721,7-benzyl-4-chloro-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidine,AGN-PC-04V4GP, MLS000562030, AGN-PC-00YPMB, T5400648, MLS003107990,AC1NUNJE, MLS002701851, SMR000185185, STK850401,[(3-bromobenzyl)sulfanyl][(4-fluorophenyl)amino]methylidene,propanedinitrile, AC1NXBLH, CAS-66-81-9, Cycloheximide, ACTIPHENOL,MLS001032885, MLS000553012, SMR000285129, MLS000688479, MLS002702480,GNF-Pf-4659, MLS002702449, T00501-4035, MLS000712179, AGN-PC-0MQWB,AGN-PC-0NKU3S, T0503-0850, T0501-5798, SMR000212173,3,3′-Diethylthiazolinocarbocyanine iodide,2-methyl-3,5-bis(4-methylphenyl)isoxazol-2-ium MLS000705900,SMR000211540, AGN-PC-00PL3I, AGN-PC-0NJNZK, SMR000354849, T0503-1204,MLS000688685, GNF-Pf-4078, T0503-3525, T0503-982, T0501-7391,GNF-Pf-3268, TCMDC-125620,1-[1,1′-Biphenyl]-4-yl-2-(4-imino-1(4H)-pyridinyl)ethanone,SMR000036350, MLS000080109, MLS000080126, Ambcb40308772, MLS000733369,Ambcb20390854, MLS000732313, AGN-PC-04RYS6, Ambcb33735952,AGN-PC-04RYKA, MLS000733096, Ambcb63657849, MLS001090213, T6132867,MLS003678910, AC1OXF5M, SMR000218920, MLS000037490, Boc-KS,MLS000734694, AGN-PC-087SDW, ISUPSL 100073, 4-{[5,7-bis(trifluoromethyl)benzenol, BAS 07204618, MLS00144057, MLS001251018, SMR000041809,SMR000635220, MLS003120011, T5546966,4-chloro-N-(4-chlorobenzyl)-1,3-dimethyl-1H-pyrazole-5-carboxamide,3-(Toluene-4-sulfonylmethyl)-2,3-dihydro-benzo[4,5]imidazo[2,1-b]thiazole,T0508-0735, Carboxyamidotriazole, MLS003116132, F0850-5968, Verrucarin A9,10-epoxide, MLS002702133, Ossamycin, MLS002702060, Dihydrorotenone,SMR000623161, Pyridaben, ASN 09858385, T6069554, T6302989, SMR000629820,SMR000629835, MLS001028777, MLS001028747, MLS001028806, SMR000625125,T5403634, T5459762, T5626573, T5337170, SMR000093473, T6120097,N-[2-[2-[2,5-dimethyl-1-(thiophen-2-ylmethyl)pyrrol-3-yl]-2-oxoethoxy]phenyl]acetamide,MLS000575323,N-[4-[2-[2,5-dimethyl-1-(thiophen-2-ylmethyl)pyrrol-3-yl]-2-oxoethyl]sulfanylphenyl]acetamide,SMR000274842, T5565081,6-chloro-N-[3-[(4-methoxyphenyl)sulfamoyl]phenyl]pyridine-3-carboxamide,N-methyl-N-[(1,3,5-trimethylpyrazol-4-yl)methyl]naphthalene-2-sulfonamide,T6099016, T6094971, ASN 04448329, SMR000241542, AGN-PC-03RL0E,AGN-PC-080KFN, T6151837, AGN-PC-0KIUAY,N-[4-(4-methylphenyl)-1,3-thiazol-2-yl]-1-thiophen-2-ylsulfonylpiperidine-4-carboxamide,5-(3,5-dimethylpiperidin-1-yl)sulfonyl-N,N-diethyl-3-methyl-1-benzofuran-2-carboxamide,SMR000124769,N-(1-benzylpiperidin-4-yl)-1-(5-chloro-2-methylphenyl)sulfonylpiperidine-4-carboxamide,MLS001095722, 4-ethoxy-N-(pyridin-4-ylmethyl)benzenesulfonamide,4-chloro-3-ethoxy-N-(pyridin-4-ylmethyl)benzenesulfonamide,2,4,6-trimethyl-N-(pyridin-4-ylmethyl)benzenesulfonamide, BAS 05598377,4-bromo-2,5-dimethyl-N-(pyridin-4-ylmethyl)benzenesulfonamide,MLS000735463, MLS000687652, AGN-PC-093SBW, AG-401/42008258, 5L-526S,2-[[5-(3-chloro-1-benzothiophen-2-yl)-1,3,4-oxadiazol-2-yl]sulfanyl]acetonitrile,2-(5-Pyridin-3-yl-[1,3,4]thiadiazol-2-ylsulfanyl)-N-quinolin-4-yl-acetamide,2-[[5-(benzotriazol-1-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]-N-[(4-chlorophenyl)methyl]-N-phenylacetamide,2-[[5-(benzotriazol-1-ylmethyl)-1,3,4-oxadiazol-2-yl]sulfanyl]-N-[(4-fluorophenyl)methyl]-N-phenylacetamide,SR-01000288264,2-(1-cyclopropyltetrazol-5-yl)sulfanyl-1-[4-[(4-propan-2-ylphenyl)methyl]piperazin-1-yl]ethanone,N-(2,4-difluorophenyl)-4-[5-(trifluoromethyl)pyridin-2-yl]-1,4-diazepane-1-carbothioamide,T0512-9975,[[2,7-bis(2-morpholin-4-ylethoxy)fluoren-9-ylidene]amino]thiourea,MLS001018548, T0507-0244,4-(4-acetylphenyl)-N-(4-phenoxyphenyl)piperazine-1-carbothioamide,N-(3-ethoxypropyl)-4-[4-(4-fluorophenyl)-1,3-thiazol-2-yl]piperazine-1-carbothioamide,(+)-Emetine dihydrochloride hydrate, MLS002302684,4-(6-chloro-1,3-benzothiazol-2-yl)-N-(2-chloro-6-methylphenyl)-1,4-diazepane-1-carboxamide,N-(3-chloro-2-methylphenyl)-4-(3-phenyl-1,2,4-thiadiazol-5-yl)-1,4-diazepane-1-carboxamide,MLS000692856, bjm-csc-19, MLS002701991, and MLS000586514, Additionalcompounds include 6-formylindole (3,2-B) carbazole,4-hydroxyphenylretinamide, 3,5-Dilodo-L-tyrosine, Rifampicin, andZ30972355.

Another aspect of the present disclosure is a pharmaceutical compositioncomprising a NR2F6 modulator, such as a NR2F6 inhibitor or NR2F6activator, for use in the methods described herein. Accordingly, incertain embodiments, the present technology provides a pharmaceuticalcomposition comprising an effective amount of a NR2F6 inhibitor or NR2F6activator in admixture with a pharmaceutically acceptable carrier,excipient or diluent. Such pharmaceutical composition can be, in variousembodiments, a composition suitable for administering to a human, or toan animal (i.e., a veterinary pharmaceutical composition).

In various embodiments, the pharmaceutical compositions herein can beused to inhibit NR2F6; or to activate NR2F6.

In certain embodiments, the pharmaceutical composition is used to treata disease or a hematopoietic condition as described herein. The NR2F6inhibitors or NR2F6 activators can be formulated into pharmaceuticalcompositions for administration to subjects in a biologically compatibleform suitable for administration in vivo.

As used herein, “biologically compatible form suitable foradministration in vivo” means a form of the substance to be administeredin which any toxic effects are outweighed by the therapeutic effects. Invarious embodiments, the substances herein can be administered to livingorganisms including humans, and animals. Administration of atherapeutically active amount of the pharmaceutical compositions of thepresent disclosure is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, atherapeutically active amount of a substance can vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of inhibitor to elicit a desired response inthe individual. Dosage regime can be adjusted to provide the optimumtherapeutic response. For example, several divided doses can beadministered daily or the dose can be proportionally reduced asindicated by the exigencies of the therapeutic situation.

In various embodiments, the active substance can be administered by,e.g., injection (subcutaneous, intravenous, intramuscular, etc.), oraladministration, inhalation, intranasal, transdermal or topicaladministration (such as topical cream or ointment, salve, paste or thelike), pulmonary, buccal, subdermal, intradermal, transdermal orparenteral, rectal, subcutaneous, intravenous, intraurethral,intramuscular, ophthalmic or suppository administration. Depending onthe route of administration, the active substance can, in certainembodiments, be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which couldinactivate the compound.

In certain embodiments, the active substance can be formulated intodelayed release formulations such that NR2F6 can be inhibited oractivated for longer periods of time than a conventional formulation.

In certain embodiments, a method herein includes the following steps:(a) extraction of an amount of a patient's cellular material, including,but not limited to: blood, saliva, sweat, or any portion of a tumorknown or believed to be in a diseased state; (b) isolating immune cellsfrom the cellular material: (c) inhibiting or activating the NR2F6target in the extracted immune cells; and (d) re-administering theimmune cells (for example, by injection) to the patient's body. This canhave the effect of “reprogramming” the immune cells to attack tumors orother invasive cells.

In certain embodiments, other types of a patient's cellular material canalso be extracted. These include, for example, any part of the blood(blood serum, red blood cells, white blood cells, plasma, platelets),any other material from the body that includes the patient's cells (forexample, skin, hair, nails, saliva, cerebrospinal fluid, intracellularfluid, extracellular fluid, intravascular fluid, interstitial fluid,lymphatic fluid, transcellular fluid, exudates, lymph, sweat, sebum orserous fluid). In certain embodiments, the re-administering of theimmune cells to the patient's body can be done by injection,introduction through the nose or mouth (for example, inhalation), skinor mucous membranes.

In certain embodiments, the present technology is directed to compoundsalone or in combination with another medicament. As set forth herein,compounds herein include stereoisomers (including, e.g., enantiomers,diastereomers, cis-trans and E-Z isomers, conformers and atropisomers),tautomers, solvates, prodrugs, metabolites, pharmaceutically acceptablesalts and mixtures thereof. Compositions containing a compound hereincan be prepared by conventional techniques, and can appear inconventional forms, for example, oral dosage forms; or any ingestible,inhalable (e.g., through the mouth, nose or mucosa); or topicalapplications, e.g., applicable to the skin, nails, eyes or the like.These can include, in various embodiments, capsules, tablets, pills,cachets, dispersible granules, lozenges, aerosols, solutions, powders,suspensions, emulsions, gels, mousses, foams, drops, lotions, creams,paste, dragees, suppositories and any application deliverable to thebody of a user.

In various embodiments, dosages and compounds herein can be prepared andadministered in a wide variety of oral, parenteral, and topical dosageforms, including, but not limited to, by injection (e.g., intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally); by inhalation (e.g., intranasally); ortransdermally. In certain embodiments, multiple routes of administrationcan be used to optimize delivery of the compounds herein.

In various embodiments, the compositions described herein can beprepared by known methods for the preparation of pharmaceuticallyacceptable compositions which can be administered to subjects, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle. On this basis, thecompositions can include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

In certain embodiments, a powder or tablet according to a dosage formherein can contain about 5 to about 75%, about 10 to about 70%, or about15 to about 65% of the active compound. Suitable carriers include, butare not limited to: magnesium carbonate, magnesium stearate, talc,sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like.

In various embodiments, carriers for certain dosages can include aqueoussolutions of dextrose, saline, water, organic solvents includingethanol, glycerol, propylene glycol, oils including peanut oil or sesameoil; or polyoxyethylene-block polymers. Aqueous solutions or suspensionscan be made by dispersing the finely divided active component in wateror another solvent with viscous material, such as natural or syntheticgums, resins, methylcellulose, sodium carboxymethylcellulose, and othersuspending agents.

In various embodiments, the compounds or dosages herein can also beincorporated into liposomes or micelles, or administered via transdermalpumps or patches.

Some compounds may have limited solubility in water and therefore mayrequire a surfactant or other appropriate co-solvent in the composition.Such co-solvents include: Polysorbate 20, 60, and 80, Pluronic F-68,F-84, and P-103; cyclodextrin: and polyoxyl 35 castor oil, and invarious embodiments, are present in amounts of about 0.01 to about 10%,about 0.05 to about 5% or about 0.1 to about 3% by weight.

In certain embodiments, it may be desirable to increase the viscosity ofthe dosage forms herein for ease in dispensing or delivery. Suchviscosity building agents include, for example, polyvinyl alcohol,polyvinyl pyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, and combinations of the foregoing; and in variousembodiments, are present in amounts of about 0.01 to about 10%, about0.05 to about 5% or about 0.1 to about 3% by weight.

The compositions herein can, in certain embodiments, additionallyinclude components to provide sustained release or comfort. Suchcomponents include, but are not limited to, high molecular weight,anionic mucomimetic polymers, gelling polysaccharides, finely-divideddrug carrier substrates, emollients, humectants, moisturizers, essentialoils, oils, lipids, fatty acids, glycerides, extracts of naturalingredients, soaps and waxes.

Useful Compounds

The present disclosure includes various compounds that were found to bemodulators of NR2F6 activity and NR2F6 utilizing compounds, and theimmune modulation and modulation of cancer stem cell activity. Exemplarycompounds and methods are shown in the attached FIGS. 1-58. Thesecompounds were initially found to be modulators of NR2F6 activity andNR2F6 utilizing compounds, and the immune modulation and modulation ofcancer stem cell activity. In various embodiments, the compoundscomprise one or more of the following functional groups: a sulfonylgroup, a sulfone group R—S(═O)2-R′ where R and R′ are any organicfunctional groups, a pyrazine group, any phenyl substituted with one ormore halogens including chlorine or fluorine; or any compositioncomprising two or more phenyl constituents. In certain embodiments, acompound herein can be in amorphous form, crystalline form, or a mixturethereof; as well as any polymorph or amorphous form, a solvate, ahydrate or an unsolvated form.

Example 1

The methodology for screening candidate compounds as NR2F6 agonists wasas follows: For primary screening, hit criteria was ACT %>DMSOcontrol+5*SD (DSO control) at 10 μM, or any compound with S/B>2. Forrescreening, hit criteria was ACT %>DMSO control+3*SD (DSO control) ineach replicate at 10 μM. For counterscreening, hit criteria was meanACT<DMSO control+3*SD (DMSO control) with ERα transient transfection induplicate at 1 μM.

Table 1 shows screening results from a first set of compounds.

TABLE 1 Firefly, Firefly_ERa, Renilla, Renilla_ERa, Compound cmpd/DMSOcmpd/DMSO cmpd/DMSO cmpd/DMSO I.D. repeat 1 repeat 2 repeat 1 repeat 2repeat 1 repeat 2 repeat 1 repeat 2 17 2.2 2.4 2.5 2.2 1.3 1.4 1.3 1.018 3.8 3.8 2.5 1.9 3.3 4.2 0.8 0.9 19 2.2 1.7 2.3 2.4 1.6 1.7 1.2 1.0 202.2 2.1 1.5 1.7 4.4 4.8 1.1 0.9 21 2.4 2.2 1.1 1.0 3.8 3.0 1.1 0.9 222.1 2.9 1.5 2.2 1.1 1.3 0.8 1.1 23 3.0 1.9 1.8 1.8 4.6 3.4 1.1 1.2 242.0 2.3 1.3 1.6 2.4 2.2 0.9 1.0 25 2.4 1.9 1.3 0.7 3.1 2.1 0.9 1.0 C13.4 4.1 1.0 1.0 4.1 1.5 1.3 1.2 C2 2.7 2.2 1.0 0.8 6.0 5.3 1.7 1.6 C32.0 2.0 1.8 1.9 1.6 1.2 1.1 1.2 C4 2.5 2.5 1.6 1.7 1.6 1.5 1.1 1.2 C52.2 1.7 1.6 2.0 1.3 1.4 1.2 1.0 C6 2.6 1.9 2.2 1.4 1.2 0.9 1.0 1.0 C72.1 2.1 0.8 0.5 1.1 1.4 0.8 0.8 C8 2.1 2.8 1.2 1.2 3.4 3.1 1.0 1.1 C92.7 1.9 2.5 2.4 2.4 2.5 0.9 1.0  C10 2.1 2.6 1.0 1.3 3.1 1.6 0.7 0.8 C11 13.5 12.6 1.7 1.7 3.8 3.3 1.3 1.2  C16 2.7 2.3 1.0 0.9 3.9 3.2 0.91.1

C1, C7 and C11 were found to have particularly good activity:

Additional compounds included the following:

Yet additional compounds tested included Compounds 17, 19, 22 and C3-C6:

Compound C11 was found to be particularly promising. FIGS. 16A and 16Bshow results of cytokins release by hPBMC and cytotox for Compound C1.For cytokines release and cytotox on hPBMCs, the Compound was tested at1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. For cytotox on HEK293.HEK293 pGL4 and HEK293 NR2F6 (full length) cmpd was tested from 50 uMwith dilution step 3.16 in duplicates.

The human PBMC were activated by 10 ng/mL PMA+500 ng/mL ionomycin. Datawere normalized to controls with (100%) w/o compounds.

Compounds related to Compound C11 were further explored, and inparticular, Compound C11 was substituted with various moieties to testhow this affected its activity.

In certain embodiments, the present technology is directed to compoundsof Formula (Ia), (Ib) or (Ic):

wherein any of R, R1 and R2 are C, H, N, O, S, a halogen, an alkylgroup, a substituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile.

In certain embodiments of any of the Formulas (I through XVII) herein,any of R, RA, RB. R1-R8, X, Q, Q1, Q2, or A can be any of the following:Me, OMe, Br, N, H, Cl, F or NO₂. In certain embodiments, any of R, RA,RB, R1-R8, X, Q, Q1, Q2, or A can be any of the following: 4-Me, 4-OMe,4-Br, 4-t-Bu, 34-di-Me, 4-Cl, 34-di-Cl, 3-Cl-4-F, 2-F, 3-Cl, 3-CH₃-4-F,4-iPr, Ph, 4-MeO—C6H4, 44-tBu, 2, 4-diMe, 2-thienyl, 2-MeO-4-Cl, 4-Cl,2-furayl, 4-F-C6H4, 2,4-diMeC6H3, 3-Me-4-F or 4-Cl-C6H4.

Compounds of Formulas I(a) and I(b) were rescreened in multiple assaysto acquire statistical confidence in the results. Results were repeatedin follow set screens from fresh powder. Analogs obtained are shown inFIG. 17B.

Four particularly useful compounds that were all based on C11 (CompoundsC12 through C15) were identified as follows:

Activity of the above compounds (C12 through C15) is shown in Table 2:

TABLE 2 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C12 20.016.7 4.5 9.4 2.6 2.9 C11 24.6 21.3 13.9 12.3 3.3 3.3 C13 14.0 12.9 6.48.2 3.2 3.2 C14 22.3 20.0 12.1 15.2 2.9 2.6 C15 0.9 1.7 5.8 10.7 1.2 1.7

Further compounds related to the Compound C11 and the compound ofFormulas (Ia) (Ib) or (Ic) were tested. These include the following:

Further results of the testing of these compounds are shown in thetables below. Each compound shows results based on the differentidentities of the R1 moiety.

For example, compounds of Formula (Ia) and (Ib) were tested withdifferent moieties as R1, and the results are shown below in Table 3:

TABLE 3 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUM- repeat repeat repeat repeat repeat repeat BER R1 1 2 1 2 1 2 C1004-CH3 2.0 1.9 1.6 1.3 1.3 1.1 C101 4-OMe 4.5 4.7 1.8 2.0 1.2 1.3 C1024-Br 13.0 15.3 5.8 6.1 2.3 2.5 C103 4-t-Bu 2.8 1.4 1.9 1.7 1.3 1.4 C1043,4-di-Me 3.1 4.4 1.7 1.8 2.3 1.2 C105 4-Cl 5.7 4.8 1.7 2.3 1.5 1.8 C1063,4-di-Cl 1.4 1.3 1.8 5.8 1.0 1.1 C107 3-Cl-4-F 11.0 11.0 8.7 6.5 1.72.1 C108 2-F 7.9 7.3 3.0 2.8 1.3 1.5 C109 3-Cl 9.2 10.3 3.4 3.3 3.5 3.4C110 3-CH3-4-F 7.6 7.2 2.3 2.1 1.7 2.0

In further embodiments, the present technology is directed to compoundsof Formula (II):

wherein any of R1 and R2 are C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile.

In certain embodiments, any of R1 and R2 can be any of the moietieslisted below in Tables 4 and 5. Various compounds of Formula (II) weretested with different moieties as R1 and R2, and the results are shownbelow in Tables 4 and 5:

TABLE 4 Firefly, Renilla., Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER R2 R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C111PhCH2CH2 H 29.8 35.9 29.4 30.8 3.7 3.9 C112 PhCH2CH2 4-CH3 11.7 10.1 5.65.1 2.5 2.8 C113 PhCH2CH2 4-F 5.8 7.0 10.2 10.8 2.2 3.2 C114 PhCH2Ch24-Cl 4.5 4.7 4.0 2.9 2.3 1.6 C115 PhCH2 H 6.8 21.9 4.8 10.9 6.2 7.2 C1164-CH3C6H4CH2 4-Cl 8.6 9.0 9.8 9.9 2.1 2.0 C117 4-OMeC6H4CH2 4-F 17.324.1 28.1 35.6 3.9 4.5 C118 4-F—C6H4CH2 4-F 13.5 17.1 11.8 14.8 2.4 2.7C119 4-F—C6H4CH2 4-Cl 18.4 16.2 27.8 27.6 2.0 2.2 C120 2-CF3C6H4 4-F 4.56.3 15.2 18.6 2.9 3.3 C121 3,4-di-MeO—C6H3 4-CH3 9.0 21.5 18.6 40.1 9.18.8 C122 3.4-di-MeO—C6H3 4-Cl 4.4 5.7 14.8 12.8 5.3 4.8 C123 2-MeO—C6H4H 17.7 16.8 31.9 31.7 8.2 10.6 C124 2-MeO—C6H4 4-F 25.5 29.8 50.0 51.46.6 6.7 C125 2-MeO—C6H4 4-Cl 28.0 24.5 30.4 33.8 2.5 2.7 C126 3-MeO—C6H44-CH3 45.1 44.8 43.5 42.2 4.8 4.3 C127 3-MeO—C6H4 4-F 36.7 56.2 43.143.5 8.3 8.0 C128 3-Cl—C6H4 4-F 1.7 5.2 1.7 10.1 4.4 5.4 C129 4-Cl—C6H44-F 0.9 1.2 1.0 0.7 3.3 4.5 C130 3.5-di-MeO—C6H3 H 2.3 3.4 2.2 6.4 6.410.2 C131 3.5-di-MeO—C6H3 4-CH3 9.5 15.3 30.8 38.2 4.8 4.7 C1323.5-di-MeO—C6H3 4-F 1.4 1.4 6.7 2.4 4.3 6.8 C133 3.5-di-MeO—C6H3 4-Cl2.1 4.3 9.9 12.6 3.2 2.6 C134 2,3-di-MeC6H3 4-CH3 38.1 31.9 21.0 18.93.7 5.4 C135 2,3-di-MeC6H3 4-F 5.6 4.9 19.8 18.6 1.9 3.1 C1362,3-di-MeC6H3 4-C1 22.1 25.8 12.5 14.9 2.9 3.0

TABLE 5 Firefly. Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER R2 R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C1594-F—C6H4 4-F 0.9 1.2 0.1 0.3 1.8 2.4 C160 2-me—C6H4 4-F 22.9 20.2 9.58.2 3.2 2.7 C161 4-Me—C6H4 H 35.4 39.0 28.6 27.7 4.0 5.1 C162 4-Me—C6H44-CH3 20.9 20.6 37.9 35.8 3.7 4.1 C163 4-Me—C6H4 4-F 2.0 5.2 6.9 15.85.5 5.0 C164 2,4-di-MeC6H3 H 29.3 19.6 13.8 8.9 3.1 2.8 C1652,4-di-MeC6H3 4-CH3 4.2 4.0 4.7 4.6 1.4 1.4 C166 2,4-di-MeC6H3 4-Cl 3.44.2 2.9 2.9 1.4 1.7 C167 3,4-di-MeC6H3 H 1.0 0.7 0.2 0.2 0.9 0.9 C1683,4-di-MeC6H3 4-CH3 11.8 11.4 14.8 14.0 2.4 1.6 C169 3,4-di-MeC6H3 4-F8.8 10.6 9.9 10.9 2.0 1.9 C170 3,4-di-MeC6H3 4-Cl 1.7 1.6 0.2 0.3 0.71.0 C171 3-MeC6H4 4-CH3 6.4 4.2 15.4 9.8 2.0 2.3 C172 3-MeC6H4 4-F 30.432.8 44.9 37.0 3.5 4.7 C173 3-MeC6H4 4-Cl 9.8 6.9 19.8 19.5 2.3 2.4 C1743,5-di-MeC6H3 H 5.8 14.3 16.0 28.3 4.3 6.0 C175 3,5-di-MeC6H3 4-CH3 13.211.9 10.5 12.0 2.5 1.9 C176 3,5-di-MeC6H3 4-F 3.7 9.6 19.0 29.1 3.9 3.8C177 4-MeOC6H4 H 17.4 8.2 6.7 4.6 1.7 1.3 C178 4-MeOC6H4 4-CH3 15.2 16.927.7 28.2 3.5 4.4 C179 4-MeOC6H4 4-F 8.7 10.1 11.9 10.5 2.4 2.5 C1804-MeOC6H4 4-Cl 34.0 34.5 30.9 30.0 2.5 2.7 C181 2-EtOC6H4 4-F 10.4 13.514.6 21.4 2.2 1.7

In further embodiments, the present technology is direct to compounds ofFormula (III):

wherein any of Q and R1 are C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile.

Various compounds of Formula (III) were tested with different moietiesas R1 and Q, and t. These include the following:

The results of the tests are shown below in Tables 6-8:

TABLE 6 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER Q R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C182Cl 3-F 16.2 17.3 15.1 13.2 2.0 2.1 C183 Br 3-F 1.2 1.2 3.1 2.4 1.1 1.2C184 Cl 3-MeO 1.1 1.4 1.2 1.8 0.9 1.1 C185 Br 3-MeO 1.6 2.4 0.1 0.3 0.81.2 C186 Cl 3,5-diMe 1.0 6.9 0,4 18.8 1.2 1.7 C187 Br 3,5-diMe 0.8 0.71.0 0.8 0.9 1.1 C188 Cl 3-Cl-4-Me 9.4 8.7 7,6 6.9 1.8 1.8 C189 Br3-Cl-4-Me 2.8 2.2 2.7 2.4 1.2 1.1 C190 F 3,5-diMe 2.1 2.5 1.4 1.4 1.31.1 C191 Cl 3-Cl 8.6 12.8 35.8 30.6 1.7 2.2 C192 Br 3-Cl 1.8 2.4 9.6 9.51.7 2.0 C193 Br 3,5-diF 1.8 1.9 6.5 14.6 1.5 1.8 C194 Cl 3,5-diF 10.66.3 7.2 6.2 1.3 1.3 C195 Cl 3-CN 38.7 29.4 22.8 10.1 3.7 5.1 C196 Br3-CN 3.8 3.4 13.6 18.8 2.3 2.9 C197 Br 4-Me 17.6 14.8 16.1 13.7 2.6 2.5C198 Br 4-F 1.0 0.8 8.8 5.3 1.2 1.1 C199 Br 4-MeO 19.3 16.1 25.9 23.22.2 2.9 C200 Br 4-t-Bu 11.0 8.1 5.8 10.4 1.6 1.9 C201 Cl H 6.1 5.3 8.88.2 1.8 2.4 C202 Cl 4-Me 15.7 14.1 8.3 8.9 2.6 2.3 C203 Cl 4-MeO 9.515.5 20.2 22.8 2.1 2.7 C204 Cl 4-Br 5.0 5.8 28.9 27.9 1.5 1.8 C205 Cl4-t-Bu 6.9 6.0 9.7 8.8 1.5 1.4 C206 MeO H 2.1 1.8 1.1 1.1 1.2 1.4 C207MeO 4-MeO 2.5 2.8 1.2 0.7 1.5 1.4 C208 MeO 4-Br 0.8 0.9 1.0 1.1 1.1 1.0C209 MeO 4-t-Bu 3.1 4.2 0.9 1.1 1.3 1.1 C210 NO2 H 22.8 14.3 15.0 9.82.4 2.1 C211 NO2 4-Me 5.8 17.7 18.1 18.9 3.3 4.0

TABLE 7 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER Q R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C137NO2 4-F 22.6 23.4 17.6 15.7 2.7 2.9 C138 NO2 4-Br 10.0 16.6 18.3 18.72.9 2.8 C139 NO2 4-t-Bu 6.6 6.4 7.0 6.7 1.4 1.8 C140 Cl 4-i-Pr 12.8 12.012.4 11.8 2.4 2.6 C141 MeO 4-i-Pr 1.6 1.6 1.2 1.3 1.4 1.1 C142 Br3,4-diMe 1.2 1.7 1.4 9.8 1.1 1.6 C143 Cl 3,4-diMe 15.2 14.5 19.0 17.72.6 2.4 C144 MeO 3,4-diMe 0.9 0.8 3.4 1.1 1.0 0.9 C145 NO2 3,4-diMe 11.224.4 20.5 20.5 3.3 3.5 C146 Br 4-Cl 1.5 0.9 0.6 3.2 0.8 1.2 C147 Cl 4-Cl2.6 2.9 1.8 5.0 1.5 1.4 C148 MeO 4-Cl 1.0 0.9 0.6 1.3 1.1 1.0 C149 NO24-Cl 7.8 15.3 15.3 19.7 2.7 2.3 C150 Br 3,4-diCl 8.5 7.9 12.2 11.5 1.71.7 C151 Cl 3,4-diCl 3.6 5.5 15.1 15.3 1.1 1.3 C152 OMe 3,4-di-Cl 4.53.1 4.4 4.2 1.3 1.1 C153 NO2 3,4-diCl 6.5 9.0 13.6 16.2 1.3 1.6 C154 Br3,4-diMeO 35.4 35.4 25.6 19.5 4.0 3.8 C155 Cl 3,4-diMeO 37.3 33.3 19.315.4 3.5 3.0 C156 MeO 3,4-diMeO 1.0 1.0 0.9 1.0 1.0 1.0 C157 NO23,4-diMeO 24.9 20.4 6.9 6.0 2.3 2.1 C158 MeO 3-Cl-4-F 1.5 1.2 2.7 2.41.1 1.2

TABLE 8 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER Q R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C212 FH 1.7 1.9 1.7 1.7 1.0 1.4 C213 F 4-F 14.0 13.6 9.0 8.1 1.2 1.7 C214 F4-MeO 1.9 1.9 1.7 1.7 1.0 1.4 C215 F 4-Br 11.6 13.8 14.8 15.1 2.1 2.2C216 F 4-t-Bu 1.3 1.0 1.3 1.0 1.3 1.2 C217 F 3-CH3-4-F 1.8 1.4 1.8 1.50.9 1.4 C218 F 4-Cl 18.2 19.1 11.9 10.2 1.9 2.0 C219 Br 2-F 1.3 2.5 8.723.5 2.0 2.3 C220 Cl 2-F 10.4 6.5 8.1 6.7 1.3 1.5 C221 MeO 2-F 1.3 0.91.3 1.3 1.4 1.7 C222 MeO 3-Cl 4.5 3.7 2.3 1.8 1.0 1.3 C223 Br 3-CH3-4-F4.5 5.1 3.7 5.9 1.9 1.4 C224 Cl 3-CH3-4-F 6.6 11.4 18.2 26.9 2.8 3.1C225 MeO 3-CH3-4-F 1.5 3.1 1.2 1.4 1.3 1.5

In further embodiments, the present technology is directed to compoundsof Formula (IV):

wherein any of Q and R1 are C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile.

Various compounds of Formula (IV) were tested with different moieties asQ and R1. These included the following:

The results are shown below in Table 9:

TABLE 9 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER Q R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C226OMe 3-OMe 1.1 1.0 0.5 1.1 1.2 1.3 C227 OMe 3,5-di-Me 1.0 1.3 1.1 1.1 1.01.1 C228 OMe 3-Cl-4-Me 1.0 1.3 2.2 2.1 1.1 1.0 C229 OMe 3,5-di-F 2.9 1.81.5 1.3 1.2 0.8 C230 OEt 3,5-di-F 12.5 8.3 4.5 3.8 1.8 1.5 C231 OMe 4-Me0.7 0.9 1.3 1.1 1.1 1.0 C232 OMe 4-OMe 1.0 0.9 1.0 1.0 1.0 0.9 C233 OMe4-Br 2.8 2.1 1.1 1.3 1.3 1.2 C234 OMe 4-t-Bu 2.2 1.8 4.3 2.7 1.0 1.1C235 OMe H 1.0 1.2 1.0 1.1 1.0 0.9 C236 OEt CH3 4.4 4.9 3.1 3.2 1.6 1.6C237 OEt 4-F 3.5 2.9 1.5 1.3 1.3 1.3 C238 OEt 4-OMe 1.3 1.4 1.5 1.0 1.31.2 C239 OEt 4-Br 1.4 2.2 8.4 17.2 1.2 1.1 C240 OEt 4-t-Bu 1.5 0.9 1.51.5 0.9 1.1 C241 OMe 4-i-Pr 2.1 2.7 1.5 1.9 1.2 1.8 C242 OMe 3,4-di-Me1.1 1.1 0.9 1.1 1.1 1.1 C243 OEt 3,4-di-Me 2.0 3.3 2.3 2.3 1.4 1.9 C244OEt 4-Cl 9.7 14.1 19.8 18.4 1.7 1.9 C245 OMe 3,4-di-Cl 1.9 1.3 1.7 1.81.1 1.3 C246 OEt 3,4-di-Cl 7.4 6.5 7.6 8.4 1.6 1.9 C247 OMe 3,4-di-OMe2.2 1.6 1.7 1.5 1.3 1.1 C248 OEt 3,4-di-OMe 8.6 4.3 2.8 1.9 1.4 1.4 C249OMe 2,5-di-OMe 1.4 1.5 1.0 1.1 1.1 1.3 C250 OMe 3-Cl-4-F 1.2 1.8 1.7 1.91.1 1.1 C251 OEt 3-Cl-4-F 2.1 1.9 2.4 2.0 1.0 0.9 C252 OEt 4-OEt 3.2 2.41.4 1.6 1.4 1.3 C253 OMe 2-F 2.5 2.7 1.5 1.2 1.3 1.2 C254 OEt 2-F 7.65.1 2.7 2.5 1.5 1.4 C255 OMe 3-Cl 1.5 1.5 1.2 1.1 1.3 1.0 C256 OEt 3-Cl11.5 7.9 5.1 3.5 2.0 2.4 C257 OMe 3-Me-4-F 2.1 1.6 1.2 1.1 0.8 1.0 C258OEt 3-Me-4-F 1.5 1.8 1.3 1.5 1.3 1.5

In further embodiments, the present technology is directed to compoundsof Formula (V):

wherein any of A and R1 are C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile.

Various compounds of Formula (V) were tested with different moieties asA and R1, and the results are shown below in Table 10:

TABLE 10 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER A R repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 C259OMe 3-OMe 1.3 1.9 1.2 1.2 0.9 1.1 C260 OMe 3,5-di-Me 1.0 1.0 1.4 1.3 1.01.1 C261 OMe 3-Cl-4-CH3 2.0 1.4 1.7 1.5 0.9 1.2 C262 OMe 3,5-di-F 1.82.2 1.4 1.2 1.4 1.3 C263 OMe 3-CN 1.8 1.7 1.4 1.3 1.2 1.0 C264 OMe H 1.10.8 1.2 1.2 0.9 1.0 C265 OMe CH3 1.0 1.3 1.4 1.3 1.2 1.1 C266 OMe OMe0.9 1.2 1.4 1.3 1.0 1.0 C267 OMe 4-Br 1.4 1.7 2.5 1.2 1.2 1.6 C268 OMe4-t-Bu 2.8 3.1 1.3 2.1 0.9 1.2 C269 OMe 4-i-Pr 3.0 3.0 1.5 1.4 1.2 1.0C270 OMe 3,4-di-Mc 3.2 3.1 2.2 1.8 1.3 1.2 C271 OMe 4-Cl 2.2 1.2 2.3 2.11.6 1.6 C272 OMe 3,4-di-Cl 4.5 3.1 4.4 4.2 1.3 1.1 C273 OH H 2.0 1.5 1.62.0 1.0 1.2 C274 OMe 3,4-di-OMe 3.3 3.5 1.2 1.5 1.6 1.2 C275 OMe3-Cl-4-F 2.6 2.5 2.3 1.9 1.2 1.3 C276 OH 3-Cl-4-F 1.2 1.3 2.1 1.9 0.91.0 C277 NEt2 4-Me 3.4 2.9 1.5 0.9 1.0 1.3 C278 NEt2 4-Br 2.4 3.0 1.51.7 1.0 1.2 C279 NEt2 4-Cl 3.9 2.1 1.4 1.6 0.9 0.9 C280 OMe 2-F 0.6 0.61.4 1.4 0.9 1.2 C281 OMe 3-Cl 3.0 4.4 1.8 1.9 1.4 1.3 C282 OMe 3-CH3-4-F0.7 1.1 1.5 1.7 1.4 2.0

In further embodiments, the present technology is directed to compoundsof Formula (VI):

wherein any of Q1, Q2 and R1 are C, H, N, O, S, a halogen, an alkylgroup, a substituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile.

Various compounds of Formula (VI) were tested with different moieties asQ1 and Q2. These included the following:

The results are shown below in Table 11:

TABLE 11 Firefly, Renilla, Firefly_pGL, ID cmpd/DMSO cmpd/DMSO cmpd/DMSONUMBER Q1 Q2 R1 repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2C283 Br Br OMe 1.0 1.3 0.1 0.2 1.0 0.9 C284 Br Br 4-i-Pr 2.9 2.5 0.4 0.30.9 1.0 C285 Br Br 3,4-di-Me 1.0 1.0 0.1 0.6 1.0 1.2 C286 Br Br 4-Cl 2.32.4 0.3 0.9 0.8 1.1 C287 Br Br 3,4-di-Cl 3.0 2.6 2.9 2.5 1.1 1.5 C288 FF H 6.8 3.3 4.4 3.3 1.6 1.4 C289 F F OMe 31.3 15.6 13.5 10.0 1.7 1.3C290 F F 4-Br 13.8 14.6 21.6 17.1 2.0 1.8 C291 F F 4-t-Bu 0.9 0.8 0.10.2 0.7 0.7 C292 F F 3-Me-4-F 18.3 14.8 15.6 13.4 1.7 1.7 C293 F F 4-Cl8.5 11.9 27.8 21.2 2.1 1.7 C294 Br OMe H 16.5 16.5 36.0 27.5 2.4 2.9C295 Br OMe 4-F 4.0 4.6 4.3 3.6 1.8 1.6 C296 Br OMe 4-OMe 6.0 7.8 14.914.1 2.2 2.0 C297 Br OMe 4-t-Bu 1.5 2.6 2.0 6.9 1.4 1.3 C298 Br OMe3-Me-4-F 2.2 3.2 1.9 1.9 1.3 1.4

In certain embodiments, the technology is directed to compounds ofFormulas (VII) or (VIII):

wherein any of X and R is: C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, and amide, an amine, an ether, a thiol or anitrile; and wherein n is an integer 1, 2, 3, 4, 5 or 6. In certainembodiments, the X—R moiety represents a benzene ring fused to then-membered ring containing the N substitution, to create a bicyclicfunctional group; see, e.g., Compounds F41 through F47 below.

Exemplary compounds in accordance with these Formulas include thefollowing:

Further exemplary compounds tested in accordance with the variousFormulas disclosed herein include the following:

Tables 12 and 13 show a summary of selected compounds for CRC analysis.

TABLE 12 Firefly/ Firefly, Renilla NR2F6 Firefly, Renilla, Firefly_pGL,(NR2F6 stable ID cmpd/DMSO cmpd/DMSO cmpd/DMSO stable, (clone NUMBERrepeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 clone F4) F4)/pGL4C289 31.3 15.6 13.5 10.0 1.7 1.3 2.0 15.3 C155 37.3 33.3 19.3 15.4 3.53.0 2.0 10.9 C157 24.9 20.4 6.9 6.0 2.3 2.1 3.5 10.2 C299 21.5 15.2 5.53.8 1.8 1.8 4.0 10.1 C218 18.2 19.1 11.9 10.2 1.9 2.0 1.7 9.5 C177 17.48.2 6.7 4.6 1.7 1.3 2.3 8.5 C164 29.3 19.6 13.8 8.9 3.1 2.8 2.1 8.2 C13622.1 25.8 12.5 14.9 2.9 3.0 1.7 8.1 C134 38.1 31.9 21.0 18.9 3.7 5.4 1.87.7 C195 38.7 29.4 22.8 10.1 3.7 5.1 2.1 7.7 C160 72.9 20.7 9.5 8.2 3.22.7 2.4 7.2 C11  24.6 21.3 13.9 12.3 3.3 3.3 1.8 7.0 C12  20.0 16.7 4.59.4 2.6 2.9 2.6 6.6 C230 12.5 8.3 4.5 3.8 1.8 1.5 2.5 6.5 C202 15.7 14.18.3 8.9 2.6 2.3 1.7 6.1 C102 13.0 15.3 5.8 6.1 2.3 2.5 2.4 5.9 C108 7.97.3 3.0 2.8 1.3 1.5 2.6 5.5 C248 8.6 4.3 2.8 1.9 1.4 1.4 2.8 4.6 C25611.5 7.9 5.1 3.5 2.0 2.4 2.3 4.5 C254 7.6 5.1 2.7 2.5 1.5 1.4 2.4 4.4C13  14.0 12.9 6.4 8.2 3.2 3.2 1.8 4.2 C112 11.7 10.1 5.6 5.1 2.5 2.82.0 4.1 C110 7.6 7.2 2.3 2.1 1.7 2.0 3.3 3.9 F312- 4.6 4.2 1.1 1.1 1.11.3 4.0 3.7 0003 C101 4.5 4.7 1.8 2.0 1.2 1.3 2.4 3.6

TABLE 13 Firefly/ Firefly, Renilla NR2F6 Firefly, Renilla, Firefly_pGL,(NR2F6 stable ID cmpd/DMSO cmpd/DMSO cmpd/DMSO stable, (clone NUMBERrepeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 clone F4) F4)/pGL4C222 4.5 3.7 2.3 1.8 1.0 1.3 2.0 3.6 C105 5.7 4.8 1.7 2.3 1.5 1.8 2.63.1 C209 3.1 4.2 0.9 1.1 1.3 1.1 3.6 3.1 C109 9.2 10.3 3.4 3.3 3.5 3.42.9 2.9 C213 14.0 13.6 9.0 8.1 1.2 1.7 1.6 9.6 C300 9.8 7.4 6.2 4.7 1.72.3 1.6 4.3 C154 35.4 35.4 25.6 19.5 4.0 3.8 1.6 9.1 C14  22.3 20.0 12.115.2 2.9 2.6 1.5 7.6 C210 22.8 14.3 15.0 9.8 2.4 2.1 1.5 8.2 C107 11.011.0 8.7 6.5 1.7 2.1 1.4 5.8 C137 22.6 23.4 17.6 15.7 2.7 2.9 1.4 8.4C161 35.4 39.0 28.6 27.7 4.0 5.1 1.3 8.1 C194 10.6 6.3 7.2 6.2 1.3 1.31.3 6.6 C188 9.4 8.7 7.6 6.9 1.8 1.8 1.2 5.0 C182 16.2 17.3 15.1 13.22.0 2.1 1.2 8.2 C200 11.0 8.1 5.8 10.4 1.6 1.9 1.2 5.4 C220 10.4 6.5 8.16.7 1.3 1.5 1.1 6.2 C118 13.5 17.1 11.8 14.8 2.4 2.7 1.1 5.9 C292 18.314.8 15.6 13.4 1.7 1.7 1.1 9.7 C180 34.0 34.5 30.9 30.0 2.5 2.7 1.1 13.1C175 13.2 11.9 10.5 12.0 2.5 1.9 1.1 5.7 C111 29.8 35.9 29.4 30.8 3.73.9 1.1 8.7 C197 17.6 14.8 16.1 13.7 2.6 2.5 1.1 6.3 C127 36.7 56.2 43.143.5 8.3 8.0 1.1 5.7 C126 45.1 44.8 43.5 42.2 4.8 4.3 1.0 9.9

TABLE 14 Firefly Renilla F4, cmpd/ pGL4, cmpd/ F4, cmpd/ Compound DMSO(mean) DMSO (mean) DMSO (mean) ID 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40uM 10 uM 2 uM 17 1.3 1.2 1.9 1.3 1.6 1.7 1.0 1.0 0.8 19 0.5 1.0 1.7 0.91.3 1.9 0.6 1.0 1.1 22 0.6 1.4 1.3 1.5 1.0 1.6 1.0 1.1 0.8 C1  0.4 0.50.5 0.6 0.7 0.7 0.2 0.2 2.1 C3  0.9 1.1 1.5 1.0 1.3 1.1 0.9 0.9 1.0 C4 0.7 0.8 1.1 1.3 0.9 1.0 0.5 0.5 0.6 C5  0.8 0.8 0.9 1.0 1.2 1.3 1.1 0.50.7 C6  0.4 0.6 1.4 1.0 1.3 1.3 0.5 0.6 0.7 C301 1.0 1.0 1.2 1.1 1.6 2.41.0 0.7 1.1 C302 0.9 0.9 1.2 0.8 1.4 1.8 1.0 1.1 0.7 C303 0.9 0.9 1.21.1 1.1 1.1 1.0 0.8 1.0 C7  0.4 0.5 0.6 0.8 0.8 0.9 0.4 0.3 1.0 C11  0.41.6 1.7 0.8 1.7 1.1 0.2 1.2 1.1 E12  0.5 0.9 0.7 1.0 0.8 0.9 1.0 0.7 0.7E53  1.4 1.1 1.0 1.6 1.4 1.5 1.1 0.8 0.9 L1  0.5 0.5 0.9 0.8 0.4 0.8 0.70.8 0.8 Z54  0.6 1.6 0.8 0.8 1.9 1.1 0.2 0.3 0.7 Z55  2.0 0.8 1.1 2.30.7 0.8 0.1 0.3 0.8 Z56  0.5 2.1 1.2 1.2 8.9 1.2 0.2 0.3 0.9 Z74  0.70.5 0.7 0.7 0.6 0.7 0.3 0.2 0.8 Z79  0.3 0.5 0.9 0.8 0.5 0.8 0.2 0.6 0.9Z81  0.4 0.7 2.4 0.7 0.9 1.4 0.3 0.3 1.1 Z83  0.7 0.6 1.2 0.5 0.9 1.00.3 0.8 1.0 Z90  0.4 2.5 0.7 0.7 2.4 0.7 0.1 0.2 0.9 Z91  1.3 1.1 1.01.1 1.0 1.0 0.8 1.1 1.1

Additional compounds were tested, including the following:

Compounds Z54, Z55 and Z56 were found to have particularly goodactivity:

Table 15 shows screening results from another set of compounds.

TABLE 15 Firefly Renilla F4, cmpd/ ERalpha F4, cmpd/ ERalpha DMSOtransient. cmpd/ DMSO transient, cmpd/ Compound (mean) DMSO (mean)(mean) DMSO (mean) ID 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM40 uM 10 uM 2 uM Z1  0.8 1.8 1.9 0.9 2.2 2.4 0.8 1.6 1.3 0.7 1.0 1.1 Z2 0.6 1.9 1.4 0.7 1.1 1.6 1.2 1.8 1.2 1.0 1.1 1.2 Z3  1.6 1.1 1.5 1.7 2.62.2 0.9 1.4 1.5 0.9 1.0 1.1 Z4  0.8 1.2 1.5 0.8 1.4 1.5 1.4 1.6 1.1 0.81.1 1.2 Z5  1.2 0.9 1.5 2.4 2.5 2.4 1.2 0.9 1.0 1.0 1.0 1.0 Z6  1.1 1.41.4 1.1 2.1 2.0 0.9 1.1 1.1 0.9 1.1 1.1 Z7  1.0 1.2 0.9 1.5 2.0 1.3 1.11.1 1.3 1.0 1.1 1.1 Z8  0.7 4.0 2.6 0.4 2.4 2.2 0.2 9.1 2.7 0.1 1.4 1.4Z9  1.2 6.7 2.5 0.5 2.7 2.2 0.3 4.9 2.1 0.2 0.8 1.3 Z10 0.7 1.6 5.8 0.51.5 4.2 0.3 3.3 3.4 0.2 0.8 1.3 Z11 0.5 1.2 3.5 0.6 1.6 2.1 0.3 2.6 2.90.2 0.8 1.4 Z12 0.4 1.8 3.9 0.4 1.3 2.4 0.4 2.7 2.7 0.2 0.7 1.3 Z13 1.60.9 1.0 1.1 1.7 1.2 0.4 0.7 0.8 0.4 0.6 0.8 Z14 1.2 0.8 1.2 1.0 1.2 1.01.0 0.9 0.8 1.0 1.1 1.1 Z15 0.9 0.5 1.0 1.1 1.1 0.9 1.0 1.1 0.8 0.9 1.01.0 Z58 0.8 4.5 0.9 0.8 6.5 1.5 0.8 1.2 1.3 0.7 1.1 1.2 Z17 0.6 6.5 1.52.9 9.5 1.7 0.6 1.1 1.2 0.6 1.1 1.3 Z61 0.8 2.5 1.7 0.7 9.1 3.0 0.6 0.51.3 0.7 0.8 1.4 Z19 0.7 6.3 1.3 0.7 12.7 1.9 0.7 0.9 1.1 0.6 1.0 1.5 Z670.7 0.5 1.3 0.5 1.4 1.4 0.6 1.0 0.6 1.0 1.1 1.1 Z68 1.2 1.1 1.5 0.7 1.51.1 0.6 1.0 0.9 0.6 0.9 1.1 Z70 1.0 1.0 1.1 1.5 1.1 0.9 1.3 1.3 0.8 1.21.0 1.1 Z71 0.8 1.0 0.8 0.4 1.0 1.4 0.4 0.9 1.1 0.5 1.0 1.0 Z75 0.8 1.20.9 0.9 1.9 1.1 0.8 1.0 1.1 0.7 0.9 1.0 Z76 0.8 1.8 0.8 0.7 1.9 1.3 0.50.8 1.0 0.4 0.8 1.1 Z78 1.6 1.5 1.4 1.1 2.4 1.5 0.8 1.0 1.1 0.6 0.9 1.0

Compounds Z8-Z12, Z17 and Z19 were found to have particularly goodactivity.

Dog's PMBC ELISA and cytotoxicity experiments were performed on CompoundZ92, which also showed good activity. Results are shown in FIGS. 19A and19B.

Another useful compound is Compound Z95:

Results of testing done on compound Z95 are shown in Table 16.

TABLE 16 NR2F6_full (stable, clone NR2F6_full F4), cmpd/DMSO(transient), cmpd/DMSO Firefly Renilla Firefly Renilla ID 40 uM 10 uM 2uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM Z95 3.7 2.2 1.20.5 0.5 1.1 2.3 1.3 1.2 0.2 0.4 1.1

FIGS. 20A and 20B show further results of cytokines release by hPBMC andcytotox on Compound Z95.

Table 17 shows screening results from another set of compounds.

TABLE 17 Firefly Renilla F4, cmpd/DMSO ERalpha transient, F4, cmpd/DMSOERalpha transient, Compound (mean) cmpd/DMSO (mean) (mean) cmpd/DMSO(mean) ID 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM2 uM Z27 0.7 1.3 1.3 0.5 2.1 1.3 0.9 1.2 1.2 0.8 1.2 1.2 Z28 1.3 0.9 1.01.3 1.1 1.8 1.2 1.2 1.0 1.0 1.3 1.1 Z29 1.1 1.1 1.3 6.7 1.2 1.0 0.5 0.90.8 0.7 1.2 0.8 Z30 1.2 1.1 1.4 1.4 1.1 1.4 0.7 1.1 1.1 1.6 1.1 1.1 Z311.1 0.9 0.8 1.3 1.6 0.9 1.0 1.0 1.1 1.0 1.0 1.0 Z32 1.5 0.9 1.8 1.5 0.81.8 1.3 1.0 1.0 0.8 1.1 1.0 Z33 6.6 2.0 1.7 8.8 1.8 2.1 0.6 1.0 1.1 0.91.5 1.0 Z34 0.7 3.8 1.6 0.9 10.3 2.0 0.6 0.8 1.2 0.7 1.0 1.5 Z35 1.2 1.51.9 7.2 2.2 2.6 0.7 2.0 1.1 0.9 1.3 1.1 Z36 0.7 1.8 0.7 5.8 1.6 1.2 0.61.8 1.0 0.8 1.6 1.2 Z37 0.7 1.4 1.0 1.2 1.5 1.1 0.5 0.9 1.0 0.7 1.7 1.0Z38 0.6 0.8 0.5 0.5 0.5 0.9 0.4 0.6 0.7 0.6 0.6 0.9 Z39 0.7 1.0 0.8 0.80.5 1.2 0.5 0.5 0.7 0.6 0.6 1.1 Z40 0.8 0.7 0.8 1.0 0.3 1.0 0.5 0.6 0.70.6 0.7 1.0 Z41 1.7 1.1 1.0 1.6 1.4 0.9 0.6 0.6 0.7 0.8 1.1 1.0 Z42 0.60.9 0.9 1.1 1.8 1.5 0.7 0.9 1.2 0.7 0.9 1.1 Z43 0.8 0.8 1.3 1.6 0.9 1.40.7 0.9 1.0 0.9 1.0 1.0 Z44 0.4 0.8 0.8 1.0 1.2 1.2 0.3 1.5 1.7 0.5 0.91.1 Z45 0.5 1.2 0.8 0.7 0.6 0.8 1.1 1.2 1.2 1.0 0.8 1.1 Z46 0.6 0.8 0.60.6 0.8 0.9 0.6 0.8 0.9 1.0 1.0 0.9 Z47 2.0 1.3 0.9 1.7 1.0 0.9 1.7 1.21.1 1.2 1.4 1.2 Z48 0.7 0.5 0.9 0.5 0.3 1.5 0.6 0.6 0.8 0.6 0.8 1.1 Z490.6 0.7 0.8 0.4 0.4 1.0 0.8 0.7 1.0 0.8 0.7 1.0 Z50 0.6 0.9 0.8 0.7 0.71.3 0.8 2.0 1.6 0.6 0.8 1.0 Z51 0.7 0.6 0.6 0.4 0.8 1.3 0.3 0.7 0.8 0.30.9 1.1 Z52 0.7 0.7 0.6 0.7 0.4 0.7 0.6 0.5 0.8 0.7 0.8 1.1 Z53 0.5 0.40.8 0.4 0.3 1.0 0.6 0.7 0.7 0.5 0.6 1.0

Compounds Z33 and Z34 were found to have particularly good activity:

Other useful compounds are Compound D28 and Compound F1:

Compound D28 was tested in a cytokine release experiment—parentcompound, dog's and human PMBC. Results are shown in FIGS. 21A-21D.

Compound Z17, previously mentioned above, was tested in a cytokinerelease experiment. Results are shown in FIGS. 22A and 22B.

Compound Z33, previously mentioned above, was tested in a cytokinerelease experiment. Results are shown in FIGS. 23A and 23B.

Another compound found to be useful is Compound E56:

FIG. 24 shows the results of testing done on Compound E56.

Additional compounds found to be useful are Compounds Z96 and Z97:

Results of testing done on compounds Z96 and Z97 are shown in Tables 18and 19, respectively.

TABLE 18 NR2F6_full (stable, clone F4), NR2F6_full (transient),cmpd/DMSO cmpd/DMSO Firefly Renilla Firefly Renilla ID 40 uM 10 uM 2 uM40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM Z96 2.6 2.6 2.9 1.21.2 1.3 2.4 2.3 2.4 0.9 1.0 1.0

TABLE 19 NR2F6_full (stable, NR2F6_full (transient), clone F4),cmpd/DMSO cmpd/DMSO Firefly Renilla Firefly Renilla ID 40 uM 10 uM 2 uM40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM Z97 1.5 2.2 2.3 1.21.1 1.1 2.3 2.8 2.4 1.1 1.1 1.0

FIGS. 25A and 25B show further results of testing done on Compound Z96.FIGS. 26A and 26B show further results of testing done on Compound Z97.

Additional compounds found to be useful are Compounds Z93 and Z94:

The results of dogs PBMC cytotox for Compounds Z93 and Z94 are includedin FIG. 19B, which also shows results for the following Compounds: Z92,E54, E55 and E53.

Additional compounds tested included the following:

Table 20 shows shows screening results from another set of compounds.

TABLE 20 Firefly Renilla F4, cmpd/DMSO ERalpha transient, F4, cmpd/DMSOERalpha transient, Compound (mean) cmpd/DMSO (mean) (mean)cmpd/DMSO(mean) ID 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40 uM 10 uM 2 uM 40uM 10 uM 2 uM 26 1.3 1.1 1.0 1.1 1.3 1.0 0.7 0.7 1.0 1.0 1.0 0.9 27 1.01.0 1.0 1.2 1.2 1.2 0.8 0.9 0.9 1.2 1.1 1.1 28 0.6 0.9 1.2 0.6 0.5 1.00.6 0.8 1.1 0.7 0.8 1.2 29 1.5 1.4 0.9 2.2 1.7 1.2 0.8 0.9 0.8 1.1 1.01.1 30 1.8 1.6 1.5 1.3 1.5 1.1 3.4 2.3 1.2 1.0 1.2 1.2 31 0.9 1.0 0.81.0 1.2 0.8 1.0 1.0 1.0 1.1 1.1 1.0 32 0.8 1.3 0.8 0.7 1.2 1.2 1.0 1.41.2 1.1 1.3 1.2 33 1.1 1.0 0.8 1.4 1.3 1.3 1.4 1.3 1.2 1.3 1.1 1.2 340.9 0.8 0.9 1.2 1.2 1.1 0.9 1.1 0.9 1.1 1.1 1.0 35 0.8 1.0 1.1 1.3 0.91.0 0.9 0.9 1.1 1.1 1.0 1.0 36 0.9 0.6 1.3 0.4 0.8 1.1 0.5 0.8 1.2 0.31.1 1.2 37 1.3 1.1 1.3 1.2 1.4 1.1 1.3 1.2 0.9 1.1 1.1 1.1 38 0.7 0.81.2 0.5 0.9 0.8 1.3 1.1 1.1 1.1 1.1 1.2 39 0.9 0.9 1.1 0.9 0.6 0.9 1.01.4 1.2 1.2 1.2 1.1 40 0.8 1.0 0.9 0.7 1.0 1.0 1.0 1.3 1.1 1.1 1.2 1.241 0.9 0.9 0.8 1.4 1.5 1.5 1.1 1.1 1.1 1.1 1.2 1.2 42 0.9 0.8 1.6 1.01.0 2.3 1.1 0.8 0.7 1.0 1.0 0.9 43 0.8 0.9 1.0 1.2 1.4 1.4 1.1 1.1 1.01.0 1.0 1.1 44 0.7 1.5 1.3 0.5 1.3 1.5 0.3 1.1 1.1 0.3 1.2 1.3 45 0.71.2 3.2 0.6 1.6 1.8 0.2 0.8 1.6 0.2 0.8 1.6

After several rounds of testing, the following compounds were found tobe particularly optimal.

Table 21 shows results of testing on Compound D28.

TABLE 21 Renilla, Firefly, cmpd/DMSO Firefly, cmpd Firefly, DMSOcmpd/DMSO Renilla, DMSO repeat repeat repeat repeat mean mean repeatrepeat mean mean 1 2 mean 1 2 plate 1 plate 2 1 2 plate 1 plate 2 14.111.7 12.8 784 644 56 55 0.7 0.5 3237 2930

FIGS. 27A and 27B show NR2F6 and LBD transient transfection,respectively, for Compound D28. Higher concentrations were excluded dueto lower signal (tox effect). FIGS. 27C and 27D show NR2F6 and LBDtransient transfection at different concentrations for differentcompounds. 9 compounds were tested on LBD transfected cells (40, 10, 2and 0.5 μM, 4 replicates). FIGS. 27E and 27F show toxicity of CompoundD28. 9 compounds were tested for cytotoxicity on LBD transfected cells(40, 10, 2 and 0.5 μM, 4 replicates). Tox effect was found to causelower signal compared to DMSO. Cytotoxicity normalized to DMSO is shownin FIG. 27F (0% cytotoxicity corresponds to DMSO signal, 100%−zerosignal).

FIGS. 28A-D show the results of a cytokind release experiment for dogand human PBMC. All compounds were tested at 5, 10, 25 and 50 uM induplicates.

Dog PBMC (1×106 cells/mL) were activated by 10 ng/mL PMA+500 ng/mLionomycin. Data were normalized to controls with (100%)/without (0%)PMA+ionomycin activation.

In further embodiments, the present technology is directed to compoundsof Formula (IX):

wherein R_(A) is C, H, N, O, S, a halogen, an alkyl group, a substitutedalkyl group, a cyclic alkyl group, an aryl group, a substituted arylgroup, a heterocyclic group, an ester, an aldehyde, a ketone, acarboxylic acid, an amide, an amine, an ether, a thiol or a nitrile. Forexample, exemplary but non-limiting compounds are shown below:

In further embodiments, the present technology is directed to compoundsof Formula (X):

wherein R_(B) is C, H, N, O, S, a halogen, an alkyl group, a substitutedalkyl group, a cyclic alkyl group, an aryl group, a substituted arylgroup, a heterocyclic group, an ester, an aldehyde, a ketone, acarboxylic acid, an amide, an amine, an ether, a thiol or a nitrile. Forexample, exemplary but non-limiting compounds are shown below:

In further embodiments, the present technology is directed to compoundsof Formula (XI):

wherein any of R₁-R₅ are C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile. For example, exemplar) but non-limiting compounds are shownbelow:

In various other embodiments, the structure of the Compounds found to beuseful have other variations. For example, in certain embodiments, thepresent technology is directed to compounds of Formula (XII):

wherein any of R₁ and R₈ is C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, or a thiol.Exemplary but non-limiting compounds are shown below:

FIGS. 29-32 show exemplary methods of formulating the compounds thathave been discussed herein.

FIG. 31A shows an exemplary synthesis of compounds including thefollowing: Compound Z17, Compound Z61, Compound Z19, Compound Z70,Compound Z71, Compound Z67. Compound Z76, Compound Z75, Compound Z78,Compound Z68, Compound Z27, Compound Z79, Compound Z64, Compound Z69,Compound Z74, Compound Z154, Compound Z80, Compound Z155, Compound Z156,Compound Z157, Compound Z158, Compound Z159.

Another compound developed herein, and found to have desirable activity,is Compound E21:

Table 22 shows results of testing on Compound E21.

TABLE 22 Firefly, DMSO Renilla Renilla, DMSO Firefly, cmpd/DMSO Firefly,cmpd mean mean cmpd/DMSO mean mean repeat 1 repeat 2 mean repeat 1repeat 2 plate 1 plate 2 repeat 1 repeat 2 plate 1 plate 2 9.5 5.6 7.3526 306 56 55 0.6 0.8 3237 2930

FIGS. 33A and 33B show NR2F6 and LBD transient transfection for CompoundE21. Higher concentrations were excluded due to lower signal (tox)effect.

FIGS. 34A and 34B show NR2F6 LBD transient transfection for CompoundE21. 9 compounds were tested on LBD transfected cells (40, 10, 2 and 0.5μM, 4 replicates). FIGS. 34C and 34D show NR2F6 and LBD transienttransfection at different concentrations for different compounds. 9compounds were tested on LBD transfected cells (40, 10, 2 and 0.5 μM, 4replicates). Tox effect was found to cause lower signal compared toDMSO. Cytotoxicity normalized to DMSO is shown in FIG. 34D (0%cytotoxicity corresponds to DMSO signal, 100%−zero signal). Allcompounds were tested at 5, 10, 25 and 50 uM in duplicates. Dog PBMC(1×106 cells/mL) were activated by 10 ng/mL PMA+500 ng/mL ionomycin.Data were normalized to controls with (100%)/without (0%) PMA+ionomycinactivation.

As shown in FIG. 36, related compounds were generated from Compound E21and tested for activity.

Additional related compounds found to have desirable activity includethe following:

Table 23 shows the screening results of certain of the above compounds.

TABLE 23 Screening Firefly Results Compound ID NR2F6 full length,cmp/DMSO (mean) F4, cmpd/DMSO (mean) Concentration, mM 40 10 2 40 10 2Compound E4 1.0 1.0 1.1 0.8 0.9 0.9 Compound E8 0.9 1.2 1.2 0.7 1.0 1.0Compound E10 1.1 1.5 1.3 0.8 1.0 1.1 Compound E15 1.1 0.8 1.2 0.8 1.11.3 Compound E57 0.9 1.2 1.3 1.3 1.3 1.0 Compound E24 1.1 1.1 1.0 0.70.9 1.0 Compound E41 0.9 1.0 1.1 0.4 0.7 0.9 Compound E52 1.4 1.3 1.00.7 0.9 0.7

Additional compounds synthesized and found to have desirable activityinclude the following:

FIG. 37 shows exemplary methods of formulating the compounds that havebeen discussed herein: specifically, exemplary synthesis of compoundsincluding the following: Compound Z160, Compound Z161, Compound Z162,Compound Z163.

As discussed above, Compound F1 was of particular interest herein:

Table 24 shows results of testing on Compound F1.

TABLE 24 Renilla, Firefly, cmpd/DMSO Firefly, cmpd Firefly, DMSOcmpd/DMSO Renilla, DMSO repeat repeat repeat repeat mean mean repeatrepeat mean mean 1 2 mean 1 2 plate 1 plate 2 1 2 plate 1 plate 2 5.64.6 5.1 310 252 56 55 1.1 1.2 32747 2930

FIGS. 38A and 38B show NR2F6 and LBD transient transfection,respectively, for Compound F1.

FIGS. 39A and 39B show NR2F6 LBD transient transfection for Compound F1.9 compounds were tested on LBD transfected cells (40, 10, 2 and 0.5 μM,4 replicates). FIGS. 39C and 39D show toxicity of NR2F6 LBD transienttransfection. 9 compounds were tested for cytotoxicity on LBDtransfected cells (40, 10, 2 and 0.5 AM, 4 replicates). Tox effectcauses lower signal compared to DMSO. Cytotoxicity normalized to DMSO isshown in FIG. 39D (0% cytotoxicity corresponds to DMSO signal, 100%−zerosignal).

FIGS. 40A-D show the results of cytokine release experiment for dogs andhuman PBMC. All compounds were tested at 5, 10, 25 and 50 uM induplicates.

Dog PBMC (1×106 cells/mL) were activated by 10 ng/mL PMA+500 ng/mLionomycin. Data were normalized to controls with (100%)/without (0%)PMA+ionomycin activation.

FIG. 41 shows the general SAR strategy for testing Compound F1 andcompounds related to it in structure. Formally, the active molecule wasdivided into four domains (Domains A through D). Each domain wasevaluated independently to establish SAR trends. Combinations ofoptimized domains evaluated additive or synergistic effect. 4 relatedanalogs were available.

For example, compounds were tested with varying values of Domain A.Exemplary compounds found to be useful are listed as follows:

In certain embodiments, a compound herein has Formula (XIII):

wherein n is an integer 1, 2, or 3, and R is any other moiety mentionedin the present disclosure (e.g., C, H, N, O, S, a halogen, an alkylgroup, a substituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile).

Similarly, compounds were tested with varying values of Domain C.Exemplary compounds found to be useful are listed as follows:

In certain embodiments, a compound herein has Formula (XIV):

wherein X is C, H, N, O, S, a halogen, an alkyl group, a substitutedalkyl group, a cyclic alkyl group, an aryl group, a substituted arylgroup, a heterocyclic group, an ester, an aldehyde, a ketone, acarboxylic acid, an amide, an amine, an ether, a thiol, a nitrile or anyother moiety mentioned herein.

In other embodiments, the present technology is directed to compoundscomprising a boronate, and synthesis of such compounds. For example,FIG. 42 shows an exemplary synthesis of a boronate compound. In variousembodiments, the synthesis achieved a yield of at least about 95%, atleast about 90% and at least about 85%; with at least about 85% purity.In various embodiments, a regioisomer was present in the yield, inamounts of about 10 to about 20%, or about 12 to about 18%. In variousembodiments, the regioisomers could be separated.

FIG. 42 shows the results of other exemplary syntheses of compoundscomprising boronate, and the relative proportions of resultantcompounds.

Another compound found to have good activity is Compound P1:

Table 25 shows results of testing on Compound P1.

TABLE 25 Renilla, Firefly, cmpd/DMSO Firefly, cmpd Firefly, DMSOcmpd/DMSO Renilla, DMSO repeat repeat repeat repeat mean mean repeatrepeat mean mean 1 2 mean 1 2 plate 1 plate 2 1 2 plate 1 plate 2 4.03.0 3.5 236 166 59 56 1.0 0.9 3030 3118

FIGS. 44A and 44B show NRdF6 and LBD transient transfection of CompoundP1.

FIGS. 45A-D show NR2F6 LBD transient transfection for 9 differentcompounds, including Compound P1.

FIGS. 46A and 46B show the results of the cytokine release experimentwith dogs PBMC.

FIGS. 47A and 47B show NR2F6 agonist activity and NR2F6 agonist activity(Renilla signal) for 7 compounds along with a DMSO control. Theconclusion is that Compound Z92 shown similar slight activity (˜3 timesfirefly signal over DMSO level) at 10 uM and 50 uM. Compound E53increases firefly activity in 6 times at 10 uM and appeared to showstrong cytotox effect (great decreasing both renilla and fireflyactivity). Both Compounds Z92 and E53 will be tested on greaterconcentration range for confirmation on both cell line with doublestable transfection (clone F1-pGL4) and cell line with transienttransfection.

In certain embodiments, the present technology is directed to compoundsof Formula (XV):

wherein R is C, H, N, O, S, an alkyl group, a substituted alkyl group, acyclic alkyl group, an aryl group, a substituted aryl group, aheterocyclic group, an ester, an aldehyde, a ketone, a carboxylic acid,an amide, an amine, an ether, a thiol or a nitrile. In certainembodiments, R is H or an alkyl group.

In certain embodiments, the present technology is directed to compoundsof Formula (XVI):

wherein any of R1 and R2 are C, H, N, O, S, a halogen, an alkyl group, asubstituted alkyl group, a cyclic alkyl group, an aryl group, asubstituted aryl group, a heterocyclic group, an ester, an aldehyde, aketone, a carboxylic acid, an amide, an amine, an ether, a thiol or anitrile. In certain embodiments, either or both of R1 and R2 are H,alkyl, phenyl, piperidine, or pyrrolidine.

Exemplary compounds include the following:

Table 26 shows activity results for two exemplary compounds, CompoundZ95 and Compound Z113.

TABLE 26 ID Firefly, cmpd/DMSO (mean) F4, cmpd/DMSO (mean) Concentration40 10 2 40 10 2 Z95 2.3 1.3 1.2 3.7 2.2 1.2 Z113 0.9 1.1 1.3 1.2 1.5 1.2

FIGS. 48-51 show embodiments of a synthetic methods of formulating acompound according to the present technology.

Further compounds found to have good activity include the following:

FIGS. 52-55 illustrate syntheses of various compounds discussed herein.

FIGS. 56A and 56B show HTS activity confirmation for various compounds.Compound E53 shown similar slight activity (˜3 times firefly signal overDMSO level) at 10 uM and 50 uM. 8010-3060 increase firefly activity in 6times at 10 uM and It seems it shown strong cytotox effect (greatdecreasing both renilla and firefly activity). Both Compound Z92 andCompound E53 will be further tested on greater concentration range forconfirmation on both cell line with double stable transfection (cloneF1-pGL4) and cell line with transient transfection.

Further compounds found to have good activity include the following:

FIGS. 57 and 58 show syntheses of exemplary compositions found to beuseful.

Table 27 shows the activity of various compounds discussed herein.

TABLE 27 Firefly, Renilla, cmpd/DMSO cmpd/DMSO ID (mean) (mean) Z134 1.10.9 Z135 1.0 1.0 Z152 1.6 1.0 Z153 2.0 1.1 Z150 1.0 0.9 Z151 1.1 1.0Z149 1.5 1.0 Z139 1.5 1.1 Z140 1.2 1.0 Z141 1.8 0.9 Z142 1.1 0.9 Z97 2.1 1.0 Z113 1.0 0.9

Further compounds found to be useful include the following:

Tables 28A and 28B, 29-29C, and 30 show the results of testing of thesecompounds.

For Tables 28A and 28B,

S/B>5 S/B>2 S/B<0.5

TABLE 28A Firefly F4, cmpd/DMSO (mean) LBD, cmpd/DMSO (mean) pGL4,cmpd/DMSO (mean) ID 40 uM 10 uM 2 uM 0.5 uM 40 uM 10 uM 2 uM 0.5 uM 40uM 10 uM 2 uM 0.5 uM D104 0.8 6.2 2.4 1.7 0.6 3.3 1.7 1.3 0.7 4.0 1.41.3 D105 0.9 5.5 2.4 1.1 0.7 3.8 1.8 1.2 0.7 3.8 1.9 1.2 D106 0.8 5.42.3 1.5 0.7 4.0 1.7 1.2 0.7 3.6 1.6 1.1 D107 4.0 2.2 1.6 1.3 2.2 2.2 1.10.9 2.5 2.0 1.5 1.2 D108 1.9 1.3 1.3 1.0 2.1 1.3 1.4 1.0 1.5 1.2 1.0 0.9D109 0.4 4.2 2.3 1.3 0.2 3.2 2.1 1.1 0.5 3.6 1.6 1.0 D110 1.4 3.5 1.91.7 0.8 3.0 1.4 1.1 0.7 3.3 1.4 1.2 D111 2.7 2.0 1.1 1.1 1.9 1.6 1.4 1.11.8 1.5 1.4 0.9 D112 2.6 2.8 1.9 1.2 1.9 1.8 1.2 1.1 2.2 2.2 1.2 1.0D113 3.3 2.8 1.6 1.3 2.5 2.2 1.3 1.1 2.0 2.6 1.1 1.2 D114 3.6 2.4 1.61.6 2.2 1.9 1.3 1.1 2.0 2.0 1.3 1.1 D115 2.2 1.4 1.1 1.0 2.2 1.3 0.9 0.92.0 1.3 1.2 1.2 D116 1.2 1.4 1.0 1.5 1.2 1.1 1.2 0.9 1.4 1.1 1.2 1.0D117 0.5 4.7 2.2 1.1 0.2 4.2 2.1 1.1 0.3 4.6 1.4 1.0 D118 2.4 6.2 1.61.6 0.4 3.8 1.8 1.2 0.4 4.8 1.5 1.2 D119 0.6 5.3 2.0 0.9 0.5 3.4 1.5 1.00.6 3.9 1.5 1.1 D120 2.2 5.1 1.5 1.3 0.8 4.1 1.7 1.1 0.8 3.7 1.5 1.4D121 1.9 4.9 1.9 0.9 0.7 3.6 1.5 1.0 0.7 3.9 1.6 1.4 D122 0.7 6.8 3.31.6 0.6 3.1 2.5 1.2 0.6 2.3 2.4 1.3 D123 0.7 7.1 2.5 1.8 0.4 4.9 1.9 1.00.6 3.4 1.7 1.2 0124 2.1 4.6 1.3 1.6 0.5 3.6 1.6 1.4 0.7 3.1 1.5 1.4D125 0.9 5.6 2.3 1.3 0.4 4.6 1.6 1.0 0.6 4.2 1.9 1.4 0126 2.3 4.5 1.81.0 2.1 2.9 1.5 1.6 0.8 3.0 1.2 1.2 D127 2.0 2.2 1.1 1.2 2.1 2.3 1.2 1.22.6 1.8 1.4 1.0 D128 3.5 2.7 1.7 1.1 2.6 2.4 1.1 1.0 1.5 2.1 1.6 1.2D129 3.4 4.3 2.0 1.2 2.4 2.7 1.7 1.1 1.6 2.9 1.5 1.4 D130 1.6 4.0 2.21.4 0.7 3.1 1.7 1.1 0.6 3.4 1.8 1.2 D131 2.3 4.1 1.9 1.4 0.7 3.6 2.0 1.30.5 2.7 1.5 1.1 D132 1.1 5.5 1.9 1.5 0.4 4.3 2.1 1.1 0.5 4.9 1.6 1.6D133 1.3 1.8 1.4 1.0 1.1 1.4 1.1 1.1 1.1 1.9 1.1 1.1

TABLE 28B Renilla F4, cmpd/DMSO (mean) LBD, cmpd/DMSO (mean) 40 10 2 0.540 10 2 0.5 ID uM uM uM uM uM uM uM uM D104 0.3 1.6 1.2 1.1 0 2 0.9 1.21.1 D105 0.7 1.7 1.2 1.1 0.4 0.9 1.1 1.0 D106 0.5 1.7 1.2 1.0 0.2 0.91.1 1.0 D107 1.5 1.2 1.0 1.1 0.7 1.1 1.1 1.1 D108 1.2 1.1 1.0 1.0 1.11.1 1.1 1.1 D109 0.5 1.6 1.2 1.1 0.3 0.9 1.0 1.0 D110 1.4 1.3 1.1 1.10.5 1.0 1.1 1.1 D111 1.4 1.1 1.0 1.0 0.8 1.1 1.1 1.1 D112 1.6 1.3 1.11.0 0.9 1.1 1.0 1.0 D113 1.4 1.3 1.0 1.1 0.7 1.1 1.0 1.1 D114 1.3 1.21.1 1.0 0.7 1.1 1.1 1.1 D115 1.1 1.0 1.0 1.0 1.1 1.1 1.1 1.1 D116 1.11.1 1.1 1.0 1.1 1.1 1.1 1.0 D117 0.4 1.7 1.3 1.0 0.1 0.9 1.1 1.0 D1181.9 1.7 1.3 1.1 0.3 1.0 1.1 1.1 D119 0.2 1.6 1.2 1.2 0.1 0.9 1.1 1.1D120 1.6 1.7 1.2 1.0 0.3 0.9 1.1 1.0 D121 1.7 1.5 1.1 1.2 0.4 1.1 1.01.1 D122 0.2 2.2 1.3 1.1 0.1 0.6 1.1 1.1 D123 0.3 1.9 1.4 1.1 0.2 0.91.1 1.1 D124 1.8 1.5 1.2 1.1 0.2 0.9 1.1 1.1 D125 1.3 1.8 1.3 1.0 0.41.0 1.1 1.1 D126 1.6 1.6 1.2 1.1 0.5 1.1 1.1 1.0 D127 1.3 1.2 1.0 1.00.9 1.1 1.1 1.2 D128 1.6 1.4 1.2 1.0 0.8 1.2 1.1 1.1 D129 1.6 1.4 1.21.1 0.6 1.0 1.1 1.0 D130 1.8 1.5 1.2 1.1 0.4 1.0 1.0 1.1 D131 1.8 1.61.2 1.1 0.4 1.1 1.1 1.1 D132 1.3 1.7 1.2 1.0 0.3 1.0 1.1 1.0 D133 1.11.1 1.0 1.0 1.0 1.1 1.1 1.0

For Tables 29A and 29B,

S/B>10|S/B>5|S/B>3|S/B<0.5

TABLE 29A Firefly Com- F4, 1% FBS, F4, 10% FBS, pGL4, 1% FBS, pGL4, 10%FBS, pound cmpd/DMSO (mean) cmpd/DMSO (mean) cmpd/DMSO (mean) cmpd/DMSO(mean) ID 40 uM 10 uM 2 uM 0.5 uM 40 uM 10 uM 2 uM 0.5 uM 40 uM 10 uM 2uM 0.5 uM 40 uM 10 uM 2 uM 0.5 uM D104 0.7 8.5 1.9 0.9 1.4 1.1 1.4 0.80.6 1.5 1.0 1.0 0.5 2.7 1.7 1.1 D104 0.4 5.1 1.6 0.9 1.0 1.3 1.5 1.3 1.02.4 1.8 1.1 0.8 2.9 1.6 1.0 D105 0.3 4.9 1.7 1.5 3.6 8.9 1.5 1.0 1.1 2.61.2 1.4 1.7 2.6 1.6 1.3 D106 0.5 4.6 1.9 0.9 1.7 7.1 1.4 1.6 0.8 2.8 1.81.1 1.2 2.7 1.2 0.9 D107 3.8 1.4 0.8 1.5 6.1 1.5 1.4 0.8 1.4 1.8 1.4 1.32.3 1.7 1.3 1.1 D134 5.4 2.0 1.2 1.0 4.6 2.1 1.0 1.1 1.4 1.3 1.3 1.0 2.01.7 1.3 1.2 D135 3.3 1.1 1.6 1.2 3.7 1.8 0.7 1.0 1.1 0.8 1.0 1.0 1.6 1.71.1 0.9 D108 1.8 1.1 1.1 1.2 2.2 1.1 1.0 1.2 1.1 1.2 1.2 1.5 1.4 1.1 1.11.1 D109 0.2 5.5 1.6 1.5 3.1 6.5 1.4 1.3 1.1 2.1 1.3 1.2 1.1 2.2 1.2 1.4D110 0.8 3.8 1.4 0.8 5.9 2.4 1.0 1.3 0.8 1.7 1.2 1.0 2.6 1.8 1.2 1.2D111 2.1 1.6 1.1 1.0 3.6 1.1 1.6 1.6 1.2 1.2 1.2 1.0 1.3 1.3 1.2 1.2D112 3.2 1.6 0.9 1.1 5.0 1.5 1.2 1.2 1.2 1.7 1.2 1.3 1.3 1.5 1.0 1.3D113 2.5 2.8 1.1 1.4 5.8 2.1 1.5 1.7 1.6 1.8 1.2 1.0 2.5 1.7 1.2 1.1D114 2.2 1.8 1.0 1.3 3.2 1.7 1.1 1.2 1.5 1.7 1.3 1.1 2.0 1.5 1.3 1.3D115 1.8 1.1 0.8 1.0 1.3 0.7 1.3 1.5 1.6 1.2 1.2 1.1 2.1 1.6 1.2 1.2D116 1.1 0.9 0.9 1.2 0.8 1.3 1.4 1.1 1.4 1.7 1.0 1.6 1.5 1.4 1.1 1.1D117 0.1 5.9 2.1 0.9 2.9 4.1 1.2 0.9 0.6 1.9 1.4 1.0 1.2 2.2 1.6 1.1D118 0.5 5.2 1.4 1.1 1.5 5.1 1.3 1.2 0.9 2.1 1.2 0.9 8.1 2.9 1.2 1.2D119 0.2 4.4 1.8 1.5 1.6 7.3 1.1 1.5 0.7 2.4 1.4 1.2 0.5 2.2 1.2 1.1D120 0.2 4.7 1.2 0.8 1.7 3.6 1.2 1.0 0.9 2.8 1.5 1.5 0.7 2.0 1.7 1.1D121 0.1 3.9 1.2 1.5 3.4 3.3 1.4 1.2 0.8 3.2 1.1 1.1 1.2 2.4 1.4 1.1D122 0.3 2.8 3.2 1.4 0.5 1.1 1.8 1.5 0.7 2.6 2.5 1.1 0.7 3.7 2.2 0.9D123 0.2 4.4 2.1 1.3 0.8 7.5 2.0 1.1 0.8 2.7 1.8 1.1 0.8 3.0 1.6 1.2D124 0.4 3.1 1.9 0.9 4.5 2.3 1.0 1.2 0.8 2.4 1.6 1.1 3.2 2.2 1.4 1.2D127 0.5 4.8 1.8 1.0 0.6 5.5 1.4 1.2 0.6 2.7 1.9 1.3 0.6 2.7 1.7 1.2D136 7.4 2.4 1.0 0.8 1.2 1.9 1.4 0.4 1.6 1.3 0.9 1.1 3.8 1.8 1.1 1.3D126 0.6 3.1 1.5 0.7 8.3 2.9 1.0 1.4 1.6 2.0 1.8 1.4 3.9 1.8 1.1 1.1D127 3.1 2.2 0.9 1.3 3.2 1.5 0.7 1.2 1.3 1.6 1.3 1.1 1.4 1.4 1.2 1.0D128 2.0 2.0 1.5 1.2 3.3 1.4 0.9 0.8 1.5 1.9 1.3 1.1 1.7 1.5 1.1 0.8D129 1.4 2.9 1.1 0.9 4.1 1.9 0.8 1.1 1.6 1.9 1.2 1.4 2.2 2.0 1.0 1.0D137 3.4 3.9 2.2 1.2 1.3 4.6 1.4 0.9 1.5 1.5 1.0 1.0 4.3 2.1 1.1 0.7D138 3.3 4.2 1.9 0.8 1.2 3.1 1.3 1.8 1.7 1.6 1.0 1.0 3.6 1.7 1.0 1.1D130 0.6 3.6 1.2 0.7 5.0 3.4 1.2 1.1 0.9 2.0 2.0 1.2 2.5 2.5 1.6 1.0D131 0.8 5.4 1.5 0.8 4.2 4.1 1.1 0.7 0.9 1.7 1.1 1.0 1.8 1.9 1.2 1.0D131 0.7 3.6 0.9 0.9 6.2 2.3 1.1 1.5 1.1 2.4 1.6 1.2 2.9 2.4 1.5 1.2D132 0.5 5.0 1.3 0.7 5.4 3.3 1.4 0.9 0.8 2.6 1.6 1.0 1.6 2.7 1.3 0.9D133 0.9 1.2 0.8 0.8 1.0 1.0 0.9 1.5 1.0 1.3 1.1 1.3 1.2 1.3 0.9 0.9

TABLE 29B Rennie F4, 1% FBS, F4, 10% FBS, Com- cmpd/DIMSO (mean)cmpd/DMSO (mean) pound 40 10 2 0.5 40 10 2 0.5 ID uM uM uM uM uM uM uMuM D104 1.0 2.4 1.4 1.1 2.2 2.5 1.3 1.0 D104 0.7 1.9 1.4 1.1 1.3 2.6 1.31.2 D105 1.6 2.0 1.2 1.1 2.6 2.1 1.2 1.1 D106 1.1 1.9 1.4 1.2 2.0 2.01.1 1.2 D107 1.8 1.3 1.2 0.9 2.1 1.3 1.0 1.0 D134 2.1 1.4 1.2 1.0 2.01.3 1.0 1.0 D135 1.7 1.2 1.3 1.0 1.9 1.1 1.1 1.0 D108 1.4 1.1 1.2 1.01.4 1.0 0.9 1.0 D109 1.1 1.7 1.3 1.0 2.4 1.9 1.2 1.0 D110 1.5 1.5 1.11.0 2.4 1.5 1.0 1.0 D111 1.7 1.3 1.1 1.1 1.8 1.2 1.1 1.1 D112 2.0 1.41.1 1.1 2.1 1.3 1.0 1.0 D113 1.8 1.6 1.2 1.3 1.8 1.2 1.0 1.0 D114 1.81.7 1.2 1.1 1.5 1.1 1.0 0.9 D115 1.5 1.2 1.0 1.1 1.3 1.1 1.0 1.0 D1161.2 1.0 1.1 1.0 1.2 0.9 0.9 0.8 D117 0.8 1.7 1.4 1.0 2.4 1.8 1.0 1.0D118 2.3 1.6 1.1 1.0 5.2 2.5 1.5 1.2 D119 0.4 1.8 1.1 1.1 1.3 2.7 1.51.4 D120 0.6 2.1 1.3 1.0 1.5 1.7 1.1 1.0 D121 1.3 1.7 1.4 1.1 2.6 1.41.1 1.0 D122 0.0 2.2 1.4 1.2 0.0 2.9 1.0 1.0 D123 0.1 1.6 1.4 1.1 0.21.9 1.1 0.9 D124 1.6 1.5 1.2 1.1 2.6 1.3 1.0 1.0 D127 1.5 1.8 1.2 1.10.4 1.7 1.1 1.0 D136 2.4 1.5 1.1 1.1 3.3 1.2 0.9 0.9 D126 1.8 1.5 1.21.2 2.8 1.7 1.1 1.0 D127 1.6 1.4 1.2 1.0 1.5 1.2 1.1 1.0 D128 1.7 1.61.1 1.2 1.5 1.1 1.0 0.9 D129 1.5 1.6 1.1 1.1 1.5 1.1 0.9 1.0 D137 3.52.1 1.3 1.0 4.0 2.1 1.4 1.2 D138 2.9 1.9 1.4 1.3 3.6 2.0 1.4 1.4 D1301.9 1.6 1.2 1.1 2.1 1.3 0.9 0.8 D131 2.0 2.0 1.3 1.0 3.0 1.8 1.1 1.0D131 1.8 1.7 1.2 1.1 2.5 1.3 1.0 0.9 D132 1.7 1.7 1.3 1.1 3.0 1.6 1.10.9 D133 1.1 1.0 1.0 1.0 1.0 1.0 0.9 0.9

For Table 29C, S/B>4 and S/B>2.

S/B>4|S/B>2

TABLE 29C Com- Firefly, F4/pGL4 for S/B(F4)>3 F4, Firefly/Renilla forS/B(F4)>3 pound 1% FBS 10% FBS 1% FBS 10% FBS ID 40 uM 10 uM 2 uM 0.5 uM40 uM 10 uM 2 uM 0.5 uM 40 uM 10 uM 2 uM 0.5 uM 40 uM 10 uM 2 uM 0.5 uMD104 5.7 4.2 3.5 4.5 D104 2.1 4.3 2.6 4.9 D105 1.9 2.1 3.4 2.5 1.4 4.3D106 1.7 2.7 2.5 3.6 D107 2.6 2.7 2.2 2.9 D134 4.0 2.3 2.6 2.3 D135 3.02.3 1.9 1.9 D108 D109 2.6 2.8 3.0 3.1 1.3 3.3 D110 2.3 2.3 2.5 2.5 D1112.7 2.0 D112 2.7 3.7 1.6 2.3 D113 2.3 3.2 D114 1.6 2.1 D115 D116 D1173.2 1.9 3.4 2.3 D118 2.5 1.8 1.8 3.2 2.8 2.1 D119 1.9 3.3 2.5 2.7 D1201.7 1.8 2.3 2.1 D121 1.2 2.9 1.4 2.3 1.3 2.4 D122 1.3 3.1 2.3 3.9 D1231.6 2.5 2.7 4.0 D124 1.3 1.4 2.0 1.7 D127 1.8 2.1 2.7 3.2 D136 4.7 3.03.1 3.5 D126 1.6 2.1 2.0 3.0 D127 2.4 2.3 1.9 2.1 D128 1.9 2.2 D129 1.82.6 D137 2.4 2.7 3.1 2.2 1.0 1.9 3.3 2.2 D138 1.9 2.7 3.2 1.8 1.1 2.23.2 1.6 D130 1.8 2.0 1.4 2.3 2.4 2.7 D131 3.3 2.3 2.2 2.7 1.4 2.2 D1311.5 2.1 2.1 2.5 D132 1.9 3.4 1.2 3.0 1.8 2.0 D133

Table 30 shows the results of further testing.

TABLE 30 Firefly ReniIla Com- F4, cmpd/DMSO (mean) LBD, cmpd/DMSO (mean)pGL4, cmpd/DMSO (mean) F4, cmpd/DMSO (mean) LBD, cmpd/DMSO (mean) pound40 10 2 0.5 40 10 2 0.5 40 10 2 0.5 40 10 2 0.5 40 10 2 0.5 ID uM uM uMuM uM uM uM uM uM uM uM uM uM uM uM uM uM uM uM uM D104 1.0 4.3 4.5 2.10.2 1.2 2.9 1.3 0.7 2.1 1.8 1.2 0.3 1.7 2.0 1.2 0.2 0.6 1.1 1.1 D134 2.82.1 2.3 1.5 1.8 1.9 1.5 1.5 1.7 1.6 1.3 1.0 1.5 1.3 1.2 1.1 0.7 1.0 0.90.9 D135 2.8 1.9 2.4 1.7 2.8 1.6 1.7 1.4 1.3 1.4 1.6 1.0 1.3 1.0 1.2 0.90.9 0.8 0.9 0.9 D136 5.2 2.7 1.1 1.5 2.9 2.6 1.5 1.0 2.6 1.8 1.0 1.7 1.41.1 0.9 0.6 1.0 1.0 1.1 1.1 D137 0.6 2.0 1.3 1.1 0.4 3.2 2.0 1.1 1.5 1.51.2 0.8 2.6 1.8 1.1 1.1 0.4 0.8 1.0 1.0 D138 1.7 2.6 1.1 1.0 0.4 3.7 1.81.3 1.5 1.5 0.9 0.9 7.3 1.2 1.0 0.8 0.3 0.9 1.1 0.9 D131 0.9 4.3 1.4 1.30.5 4.3 1.7 0.8 1.0 2.3 1.3 1.0 0.3 1.9 1.0 1.0 0.2 0.8 1.1 1.1

For Table 30, S/B>4 and S/B>2. S/B>4 S/B>2

Table 31 shows further results of testing.

TABLE 31 ID Activity, renilla, NUMBER cmpd/DMSO cmpd/DMSO D104 7.1 2.2D105 5.7 1.5 D106 5.2 2.2 D109 6.2 2.1 D122 6.6 2.0 D123 6.1 2.4 D1257.3 2.5 D132 6.6 2.1

For Table 31,

S/B>5

FIGS. 59A-D show results of testing of Compounds D104, D118, D122 andD137. Compounds were tested at 10% FBS.

Table 30 shows the results of further testing on firely and renilla. ForTable 30,

S/B>4 S/B>2

Compound D136 was further tested, and results are shown in FIGS. 60A-C.For cytokines release and cytotox on hPBMCs each compound was tested at1.25, 2.5, 5, 10, 25 and 50 uM in duplicates. Human PBMC were activatedby 10 ng/mL PMA+500 ng/mL ionomycin. Data were normalized to controlswith (100%) w/o cmpds.

FIGS. 60B and 60C show comparisons with other compounds, includingCompounds 140, 141, 142 and 166.

FIGS. 61A-61E show further results of cytokine release by hPBMC forCompound D136. Each compound was tested at 1.25, 2.5, 5, 10, 25 and 50uM in duplicates.

Human PBMC were activated by 10 ng/mL PMA+500 ng/mL ionomycin. Data werenormalized to controls with (100%) w/o cmpds.

No IL1b release activation by hPBMC was observed at high releaseactivation of others cytokines.

FIGS. 62A and 62B show stability in plasma (human plasma and rat plasma)and various pH solubility data for Compound D136.

Table 33 shows the pH test results for this compound.

TABLE 33 pH 2 pH 4 pH7 OD, solubility, mg/ml solubility, mg/mlsolubility, mg/ml ID nm value SD value SD value SD D136 250 0.66 0.010.22 0.06 0.19 0.01

FIGS. 63A and 63B show stability in simulated gastric fluid (SGF) andsimulated intestinal fluid (SIF), respectively, for Compound D136.

FIGS. 64A and 64B show microsomal stability in human liver microsomesand rat liver microsomes, respectively, for Compound D136.

Tables 34 and 35 show CYP and PPB data for Compound D136.

TABLE 34 Control inhibitor final Test concen- CYP com- tration, IC50IC50 isoform pound uM M uM 1A2- D136 10-0.0098 uM >1E−05 >10 noPhenacetin inhibition 2C19- D136 100-0,098 uM >1E−05 >10 no Mephenytoininhibition 3A4- D136 100-0.098 uM >1E−05 >10 no Midazolam inhibition3A4- D136 100-0.098 uM >1E−05 >10 no Testosterone inhibition 2D6-Dextro-D136 100-0.098 uM >1E−05 >10 no methorphan inhibition 2C9- D136100-0.098 uM >1E−05 >10 no Tolbutamide inhibition 2C8- D136 100-0.098uM >1E−05 >10 no Amodiaquine inhibition

TABLE 35 %, Free Permeability, Recovery %, Free (recalc from %, Con-Species Cmpd % % (50% pl) 50% pl) Bound clusion Human D136 94 56.9 65.348.4 51.6 Low Rat D136 94 122 64.9 48.0 52.0 Low

FIGS. 65A and 65B show PK in rat plasma for Compound D136.

FIGS. 66A and 66B show PK in mice plasma for Compound D136.

Tables 36, 37 and 38 show related data.

TABLE 36 Parameter Units Estimate K_el 1/h 0.75 T_1/2 h 0.93 Tmax h 0.5Cmax ng/ml 165 AUClast h*ng/ml 197 AUCINF h*ng/ml 208 Vz/F ml/kg 66315Cl/F ml/h/kg 51948 MRTlast h 1.13 MRTINF h 1.33

TABLE 37 PO plasma IV plasma AUCinf Dose, AUCinf Dose, AUV last mg/kgAUV last mg/kg Fabs, % 208 7.53 242 1.13 12.9% 197 7.53 239 1.13 12.3%

TABLE 38 Parameter Units Estimate K_el 1/h 2.11 T_1/2 h 0.33 Tmax h0.083 Cmax ng/ml 524 AUClast h*ng/ml 239 AUCINF h*ng/ml 242 Vz/F ml/kg2263 Cl/F ml/h/kg 4814 MRTlast h 0.35 MRTINF h 0.37

Tables 39, 40 and 41 show related data.

TABLE 39 Parameter Units Estimate K_el 1/h 1.20 T_1/2 h 0.58 Tmax h 0.25Cmax ng/ml 89.6 AUClast h*ng/ml 116 AUCINF h*ng/ml 117 Vz/F ml/kg 22544Cl/F ml/h/kg 27092 MRTlast h 1.04 MRTINF h 1.08

TABLE 40 PO plasma IV plasma AUCinf Dose, AUCinf Dose, AUV last mg/kgAUV last mg/kg Fabs, % 117 3.18 124 1.12 33.2% 116 3.18 124 1.12 33.0%

TABLE 41 Parameter Units Estimate K_el 1/h 1.16 T_1/2 h 0.60 Tmax h0.083 Cmax ng/ml 390 C0 ng/ml 770 AUClast h*ng/ml 124 AUCINF h*ng/ml 124Vz ml/kg 7743 Cl ml/h/kg 9006 MRTlast h 0.25 MRTINF h 0.27 Vss ml/kg2413

A Caco-2 permeability assay was performed on Compound D136. Results areshow in table 42.

TABLE 42 P_(app) A-B, P_(app) B-A, Asymmetry ID 10⁻⁶ cm/s 10⁻⁶ cm/sIndex Compound 33.6 47.9 D136 36.0 54.6 33.1 50.9 Mean 34.2 51.1 1.5 SD1.5 3.4 CV 4.5 6.6

Regarding Compound D136, the following were key characteristics:

1. Activates full-length and ligand binding domain-only NR2F6 incellular assays.

2. Selectively inhibits IL-1, TNF-α, and IL-17a in human PBMC.

3. Stable in human and rat plasma with low PPB.

4. Extremely stable in the presence of human, rat and mouse livermicrosomes and no inhibition of 7 major isoforms of hCyt P450.

5. PK in rat, mouse and dog completed—orally bioavailable (˜33%absorbance).

6. MTD and acute toxicity in mice completed—no toxicity was observed.

7. Solubility and pH stability studies completed—soluble at pH2, pH4 andpH7.4, extremely stable at pH2 and pH7.

8. Caco-2 permeability and asymmetry—highly permeable and not asubstrate for P-gp (MDR1).

9. Considered a Class II biopharmaceutical.

The present technology is directed to a composition according to any ofthe compounds described herein, substantially free of impurities. Asused herein, the term “substantially free of impurities” means that thecompound contains no significant amount of extraneous matter, includingstarting materials, residual solvents, or any other impurities that mayresult from the preparation or isolation of the compounds herein. Invarious embodiments, at least about 95%, at least about 97% or at leastabout 98% by weight of a compound herein is present in a dosage formherein.

Although the present technology has been described in relation toparticular embodiments thereof, these embodiments and examples aremerely exemplary and not intended to be limiting. Many other variationsand modifications and other uses will become apparent to those skilledin the art. The present technology should, therefore, not be limited bythe specific disclosure herein, and can be embodied in other forms notexplicitly described here, without departing from the spirit thereof.

1. A method of increasing activity of a cell in an animal, comprisingcontacting the cell with a compound, or pharmaceutically acceptable saltthereof, selected from the group consisting of:


2. A method of reducing the size of a tumor in an animal, comprisingadministering to the tumor a compound, or pharmaceutically acceptablesalt thereof, selected from the group consisting of:


3. The method of claim 2, comprising the steps of: (a) extracting anamount of an animal's cellular material; (b) isolating immune cells fromthe cellular material; (c) activating the NR2F6 target in the isolatedimmune cells by binding them with the compound, or pharmaceuticallyacceptable salt thereof; and (d) re-administering the isolated immunecells to the animal's body.
 4. The method of claim 3, wherein theanimal's cellular material is blood.
 5. The method of claim 3, whereinthe re-administering step comprises injection of the immune cells intothe animal's body.
 6. A method of treating or reducing the effect of areaction, disease or disorder, the method comprising activating theNR2F6 target in immune cells of an animal by contacting them with acompound, or pharmaceutically acceptable salt thereof, selected from thegroup consisting of:


7. The method of claim 6, wherein the reaction, disease or disordercomprises an autoimmune response.
 8. A method of treating a disorder inan animal comprising administering to a subject an effective amount of acompound, or pharmaceutically acceptable salt thereof, selected from thegroup consisting of:


9. The method of claim 8, wherein the disorder is cancer.